SIRT2 regulates tumour hypoxia response by promoting HIF-1α hydroxylation.

Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that has a central role in the regulation of tumour metabolism under hypoxic conditions. HIF-1α stimulates glycolytic energy production and promotes tumour growth. Sirtuins are NAD(+)-dependent protein deacetylases that regulate cellular metabolism in response to stress; however, their involvement in the hypoxic response remains unclear. In this study, it is shown that SIRT2-mediated deacetylation of HIF-1α regulates its stability in tumour cells. SIRT2 overexpression destabilized HIF-1α under hypoxic conditions, whereas HIF-1α protein levels were high in SIRT2-deficient cells. SIRT2 directly interacted with HIF-1α and deacetylated Lys709 of HIF-1α. Deacetylation of HIF-1α by SIRT2 resulted in increased binding affinity for prolyl hydroxylase 2, a key regulator of HIF-1α stability, and increased HIF-1α hydroxylation and ubiquitination. Moreover, a pharmacological agent that increased the intracellular NAD(+)/NADH ratio led to the degradation of HIF-1α by increasing SIRT2-mediated deacetylation and subsequent hydroxylation. These findings suggest that SIRT2-mediated HIF-1α deacetylation is critical for the destablization of HIF-1α and the hypoxic response of tumour cells.

Docosahexaenoic acid induces the degradation of HPV E6/E7 oncoproteins by activating the ubiquitin-proteasome system.

The oncogenic human papillomavirus (HPV) E6/E7 proteins are essential for the onset and maintenance of HPV-associated malignancies. Here, we report that activation of the cellular ubiquitin-proteasome system (UPS) by the omega-3 fatty acid, docosahexaenoic acid (DHA), leads to proteasome-mediated degradation of E6/E7 viral proteins and the induction of apoptosis in HPV-infected cancer cells. The increases in UPS activity and degradation of E6/E7 oncoproteins were associated with DHA-induced overproduction of mitochondrial reactive oxygen species (ROS). Exogenous oxidative stress and pharmacological induction of mitochondrial ROS showed effects similar to those of DHA, and inhibition of ROS production abolished UPS activation, E6/E7 viral protein destabilization, and apoptosis. These findings identify a novel role for DHA in the regulation of UPS and viral proteins, and provide evidence for the use of DHA as a mechanistically unique anticancer agent for the chemoprevention and treatment of HPV-associated tumors.

TATA element-binding protein is important to epidermal growth factor-dependent induction of H2B histone gene expression in primary hepatocytes from rat.

Epidermal growth factor (EGF) is a potent mitogen for rat hepatocytes and mammalian histone synthesis is functionally and temporally coupled to DNA replication. To gain an insight on the role of EGF in the regulation of H2B histone gene expression in primary hepatocyte cultures, the binding patterns of nuclear proteins to various elements in the H2B histone gene upstream region have been investigated. EGF induced H2B histone mRNA with maximal stimulation reached at 36 hours. The induction of H2B histone mRNA was dependent on the concentration of EGF and almost reduced by actinomycin-D pretreatment. In DNase I footprinting analysis, one nuclear factor (TATA element-binding protein, TBP) bound at -20 bp (TATA element) in either the absence or presence of EGF. One DNA-protein complex was formed by DNA mobility shift assay when TATA element was incubated with nuclear extract prepared from EGF-free hepatocytes, and the amount of TBP was increased after EGF treatment. These results suggest that TBP may be correlated with transcriptional regulation of H2B histone gene by EGF in primary hepatocytes.

Glucocorticoid regulation of androgen binding protein expression in primary Sertoli cell cultures from rats.

Glucocorticoids are known to inhibit testicular function, and its receptor is also localized in the Sertoli cells. To evaluate possible role of glucocorticoid in Sertoli cells, the effects of dexamethasone on the expression of androgen binding protein (ABP) have been investigated in primary Sertoli cell cultures. Dexamethasone increased ABP mRNA levels, with maximal stimulation reached at 36 hr. The induction of ABP mRNA was dependent on the low concentration (10(-8) and 10(-7) M) of dexamethasone but gradually reduced in the cells treated with high concentration (10(-6) and 10(-5) M). Dexamethasone-induced ABP mRNA level was no change in the cells after addition of cycloheximide but almost reduced by actinomycin-D pretreatment. Steady-state levels of ABP mRNA gradually increased in the Sertoli cells prepared from 14- and 21-days of age corresponding to rat puberty, and ABP mRNA was induced by dexamethasone. These results suggest that ABP gene is transcriptionally regulated by dexamethasone in primary Sertoli cell cultures.

Promotion of microtubule assembly in vitro by a novel 35-kDa protein purified from human term placenta.

Microtubule assembly promoting-protein (taxol-like protein, TALP) was purified by combination of high salt extraction, phosphocellulose chromatography and hydroxyapatite chromatography from human term placenta. Molecular weight of purified protein was identified as 35kDa on SDS-polyacrylamide gel electrophoresis. In vitro, TALP promoted microtubule assembly in dose-dependent manner in spite of the absence of GTP. Both TALP (0.5 microM) and taxol (10 microM) gave hyperbolic kinetics and shortened the lag time for microtubule polymerization. TALP-stabilized microtubules were resistant to depolymerization by cold (4 degrees C) and CaCl2 (4 mM) like taxol-stabilized microtubules. TALP showed its direct binding on the microtubule in cosedimentation assay. These results suggest that the action mechanism of TALP on the microtubule assembly is similar to taxol in vitro and the binding site of TALP is available on the intact microtubule.

Acylshikonin analogues: synthesis and inhibition of DNA topoisomerase-I.

Compounds bearing an acyl group of a various size at 1'-OH of shikonin were synthesized as acyl analogues of shikonin, which was isolated from the root of Lithospermum erythrorhizon, and evaluated for inhibitory effect on topoisomerase-I activity. A selective acylation at 1'-OH of shikonin in the presence of dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine gave rise to a good yield of corresponding acylshikonin derivatives. In general, analogues with an acyl group of shorter chain lengths (C2-C6) exerted a stronger inhibitory action than those with longer chain lengths (C7-C20). While the halogen substitution at C-2 of the acetyl moiety failed to increase the inhibitory potency, the placement of double bonds in the acyl group (C5-C7) augmented the potency remarkably. Of the 32 derivatives evaluated, 15 compounds exhibited a higher inhibitory effect than shikonin. Noteworthy, the inhibitory potency of acetylshikonin, propanoylshikonin, and 4-pentenoylshikonin was approximately 4-fold greater than that of camptothecin. All these data suggest that the size of acyl moiety is important for the enhancement of potency, and the presence of olefinic double bonds is also beneficial.

Testosterone regulation of proto-oncogene c-myc expression in primary Sertoli cell cultures from prepubertal rats.

The expression of c-myc has been associated with cell proliferation through changes of nuclear function. To evaluate the possibility that the proto-oncogene c-myc plays a role in testosterone-dependent gene regulation, the effects of testosterone on the expression of c-myc have been investigated in primary Sertoli cell cultures. Testosterone increased c-myc mRNA levels, with maximal stimulation reached in 16 hours. The induction of c-myc mRNA was dependent on the concentration of testosterone. Testosterone-induced c-myc mRNA levels were also increased in cells after addition of cycloheximide but reduced by actinomycin-D pretreatment. Even in the absence of hormone in culture medium, c-myc mRNA was clearly detectable in Sertoli cells from 8-day-old rats but hardly detectable in cells from 14 and 28 days of age. Testosterone stimulated c-myc mRNA expression in the Sertoli cells from only 8-/and 14-day-old rats. These results suggest that testosterone induces c-myc mRNA levels in the primary Sertoli cells from prepubertal rats, and then transient expression of c-myc may be responsible for some of the regulatory roles of testosterone-dependent genes in the Sertoli cells. The biological significance of testosterone-dependent c-myc induction is not known.

Regenerating liver-specific transacting factors of H2B histone gene are newly synthesized during liver regeneration.

We have investigated DNA synthesis and levels of H2B histone mRNA, and the binding pattern of nuclear proteins to various elements in the rat H2B histone gene upstream region with DNase I footprinting assay. Both DNA synthesis and H2B histone mRNA level were increased with maximal stimulation reached at 24 hrs and 36 hrs after partial hepatectomy, respectively. In DNase I footprinting analysis, the nuclear factors interacting with the three elements, TATA at -19 bp (site AR), site B at -29 bp, and CAAT at -69 bp (site C) were required during maximal increase of H2B histone mRNA level after partial hepatectomy. The DNase I protection pattern by nuclear extract of the cycloheximide-treated regenerating liver showed the same results with normal liver. These results suggest that transcriptional regulation of H2B histone gene during liver regeneration may be mediated by nuclear factors that are newly induced by partial hepatectomy.