Sirt1 inhibitor, Sirtinol, induces senescence-like growth arrest with attenuated Ras-MAPK signaling in human cancer cells.

The induction of senescence-like growth arrest has emerged as a putative contributor to the anticancer effects of chemotherapeutic agents. Clinical trials are underway to evaluate the efficacy of inhibitors for class I and II histone deacetylases to treat malignancies. However, a potential antiproliferative effect of inhibitor for Sirt1, which is an NAD(+)-dependent deacetylase and belongs to class III histone deacetylases, has not yet been explored. Here, we show that Sirt1 inhibitor, Sirtinol, induced senescence-like growth arrest characterized by induction of senescence-associated beta-galactosidase activity and increased expression of plasminogen activator inhibitor 1 in human breast cancer MCF-7 cells and lung cancer H1299 cells. Sirtinol-induced senescence-like growth arrest was accompanied by impaired activation of mitogen-activated protein kinase (MAPK) pathways, namely, extracellular-regulated protein kinase, c-jun N-terminal kinase and p38 MAPK, in response to epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I). Active Ras was reduced in Sirtinol-treated senescent cells compared with untreated cells. However, tyrosine phosphorylation of the receptors for EGF and IGF-I and Akt/PKB activation were unaltered by Sirtinol treatment. These results suggest that inhibitors for Sirt1 may have anticancer potential, and that impaired activation of Ras-MAPK pathway might take part in a senescence-like growth arrest program induced by Sirtinol.

Japanese fermented soybean food as the major determinant of the large geographic difference in circulating levels of vitamin K2: possible implications for hip-fracture risk.

Increasing evidence indicates a significant role for vitamin K in bone metabolism and osteoporosis. In this study, we found a large geographic difference in serum vitamin K2 (menaquinone-7; MK-7) levels in postmenopausal women. Serum MK-7 concentrations were 5.26 +/- 6.13 ng/mL (mean +/- SD) in Japanese women in Tokyo, 1.22 +/- 1.85 in Japanese women in Hiroshima, and 0.37 +/- 0.20 in British women. We investigated the effect of Japanese fermented soybean food, natto, on serum vitamin K levels. Natto contains a large amount of MK-7 and is eaten frequently in eastern (Tokyo) but seldom in western (Hiroshima) Japan. Serum concentrations of MK-7 were significantly higher in frequent natto eaters, and natto intake resulted in a marked, sustained increase in serum MK-7 concentration. We analyzed the relation between the regional difference in natto intake and fracture incidence. A statistically significant inverse correlation was found between incidence of hip fractures in women and natto consumption in each prefecture throughout Japan. These findings indicate that the large geographic difference in MK-7 levels may be ascribed, at least in part, to natto intake and suggest the possibility that higher MK-7 level resulting from natto consumption may contribute to the relatively lower fracture risk in Japanese women.

Long-term denervation impairs insulin receptor substrate-1-mediated insulin signaling in skeletal muscle.

Long-term denervation is associated with insulin resistance. To investigate the molecular bases of insulin resistance, the downstream signaling molecules of insulin receptor including insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI 3-K) were examined in skeletal muscle of rats after 7 days of denervation. Long-term denervation attenuated insulin-stimulated activation of the initial steps of the intracellular signaling pathway. Insulin-stimulated tyrosine phosphorylation of insulin receptor was reduced to 36% (P < .005), as was the phosphorylation of IRS-1 to 34% (P < .0001) of control. While insulin receptor protein level was unchanged, the protein expression of IRS-1 was significantly decreased in denervated muscles. Insulin-stimulated percent tyrosine phosphorylation of IRS-1, normalized to the IRS-1 protein expression, was also reduced to 55% (P < .01) of control in denervated muscle. Denervation caused a decline in the insulin-induced binding of p85 regulatory subunit of PI 3-K to IRS-1 to 61% (P < .001) and IRS-1-associated PI 3-K activity to 57% (P < .01). These results provide evidence that long-term denervation results in insulin resistance because of derangements at multiple points, including tyrosine phosphorylation of insulin receptor and its downstream signaling molecule, IRS-1, protein expression of IRS-1, and activation of PI 3-K.

Immobilization depresses insulin signaling in skeletal muscle.

Prolonged immobilization depresses insulin-induced glucose transport in skeletal muscle and leads to a catabolic state in the affected areas, with resultant muscle wasting. To elucidate the altered intracellular mechanisms involved in the insulin resistance, we examined insulin-stimulated tyrosine phosphorylation of the insulin receptor beta-subunit (IR-beta) and insulin receptor substrate (IRS)-1 and activation of its further downstream molecule, phosphatidylinositol 3-kinase (PI 3-K), after unilateral hindlimb immobilization in the rat. The contralateral hindlimb served as control. After 7 days of immobilization of the rat, insulin was injected into the portal vein, and tibialis anterior muscles on both sides were extracted. Immobilization reduced insulin-stimulated tyrosine phosphorylation of IR-beta and IRS-1. Insulin-stimulated binding of IRS-1 to p85, the regulatory subunit of PI 3-K, and IRS-1-associated PI 3-K activity were also decreased in the immobilized hindlimb. Although IR-beta and p85 protein levels were unchanged, IRS-1 protein expression was downregulated by immobilization. Thus prolonged immobilization may cause depression of insulin-stimulated glucose transport in skeletal muscle by altering insulin action at multiple points, including the tyrosine phosphorylation, protein expression, and activation of essential components of insulin signaling pathways.

Phorbol ester-induced generation of reactive oxygen species is protein kinase cbeta -dependent and required for SAPK activation.

Treatment of human U-937 myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with protein kinase C (PKC) betaII-mediated activation of the stress-activated protein kinase (SAPK) pathway. The present studies demonstrate that the TPA response of U-937 cells includes the generation of reactive oxygen species (ROS). By contrast, the TPA-resistant U-937 cell variant (TUR), which is deficient in PKCbetaII expression, failed to respond to TPA with the induction of ROS. Moreover, we show that TPA-induced ROS production is restored in TUR cells stably transfected to express PKCbetaII. The results also demonstrate that TPA-induced ROS production is required for activation of the MEK kinase-1 (MEKK-1)--> SAPK pathway. In concert with this observation, treatment of U-937 with H(2)O(2) as a source of ROS is associated with activation of the MEKK-1-->SAPK cascade. These findings indicate that PKCbetaII is required for TPA-induced ROS production and that the MEKK-1-->SAPK pathway is activated by a ROS-mediated mechanism.

Skeletal muscle apoptosis after burns is associated with activation of proapoptotic signals.

Critical illness is associated with muscle wasting and muscle weakness. Using burn injury as a model of local and systemic inflammatory response, we tested the hypothesis that thermal injury causes apoptosis in muscle. After a 40% body surface area burn to rats, abdominal muscles beneath the burn and limb muscles distant from the burn were examined for apoptosis at varying times after burn. Ladder assay, ELISA, and histological methods showed evidence of apoptosis in the abdominal muscles within 4-12 h with peak changes occurring at 3-7 days. Maximal apoptosis was also evident at distant limb muscles at 3-7 days. Investigation of proapoptotic pathways indicated mitochondrial membrane potential to be altered by 1 h after burn. Starting at 15 min after burn, cytochrome c was released from the mitochondria into the cytosol, followed by increased activity of caspase-3, starting at 6 h after burn. These studies suggest that mitochondria and caspase-mediated apoptotic pathways may be an additional mechanism of muscle weight loss in burns and may be potential therapeutic targets for prevention of muscle wasting.

Activation of MEK kinase 1 by the c-Abl protein tyrosine kinase in response to DNA damage.

The c-Abl protein tyrosine kinase is activated by certain DNA-damaging agents and regulates induction of the stress-activated c-Jun N-terminal protein kinase (SAPK). Here we show that nuclear c-Abl associates with MEK kinase 1 (MEKK-1), an upstream effector of the SEK1-->SAPK pathway, in the response of cells to genotoxic stress. The results demonstrate that the nuclear c-Abl binds to MEKK-1 and that c-Abl phosphorylates MEKK-1 in vitro and in vivo. Transient-transfection studies with wild-type and kinase-inactive c-Abl demonstrate c-Abl kinase-dependent activation of MEKK-1. Moreover, c-Abl activates MEKK-1 in vitro and in response to DNA damage. The results also demonstrate that c-Abl induces MEKK-1-mediated phosphorylation and activation of SEK1-SAPK in coupled kinase assays. These findings indicate that c-Abl functions upstream of MEKK-1-dependent activation of SAPK in the response to genotoxic stress.

Translocation of SAPK/JNK to mitochondria and interaction with Bcl-x(L) in response to DNA damage.

Activation of the stress-activated protein kinase (SAPK/JNK) by genotoxic agents is necessary for induction of apoptosis. We report here that ionizing radiation ionizing radiation exposure induces translocation of SAPK to mitochondria and association of SAPK with the anti-apoptotic Bcl-x(L) protein. SAPK phosphorylates Bcl-x(L) on threonine 47 (Thr-47) and threonine 115 (Thr-115) in vitro and in vivo. In contrast to wild-type Bcl-x(L), a mutant Bcl-x(L) with the two threonines substituted by alanines (Ala-47, Ala-115) is a more potent inhibitor of ionizing radiation-induced apoptosis. These findings indicate that translocation of SAPK to mitochondria is functionally important for interactions with Bcl-x(L) in the apoptotic response to genotoxic stress.

The 1999 Moyer award. Burn injury induces skeletal muscle apoptosis and the activation of caspase pathways in rats.

Burn injury induces many metabolic disorders, including altered protein kinetics with muscle weakness. The skeletal muscle weakness that occurs as a result of the loss of muscle mass causes hypoventilation and dependence on respirators, a condition that increases morbidity and mortality. The presence or absence of apoptosis in muscle, which can be a cause of the loss of muscle mass, was studied in rats after they had received scald burns to 40% of their body surface areas. The potential pro-apoptotic pathways that were activated were also examined. The burn injury produced did not directly destroy the muscle beneath; muscles just beneath the burned surface showed dramatic apoptotic changes according to assessments with the cell death enzyme-linked immunosorbent assay and in situ TdT-mediated dUTP-X nick-end labeling staining. The extent of apoptosis reached a peak on postburn days 3 and 7. Of note is that apoptosis was also confirmed in muscles at sites distant from the burn injury (eg, tibialis anterior) on both postburn days 3 and 7, a condition that is suggestive of the systemic effects of pro-apoptotic factors. To show that heat itself causes the initiation of the pro-apoptotic signaling, muscle-derived C2C12 cells were subjected to heat treatment at 55 degrees C. Ceramide, a key apoptotic second messenger, was observed to increase in the caveolae fraction but not in non-caveolae fraction of these muscle cells. In muscle tissue from burned rats, stress-activated protein kinase (a downstream-signaling kinase of ceramide) was activated soon after burn injury; this finding is consistent with the hypothesis that ceramide plays a role in burn-induced apoptosis. Caspase-1, -3, and -9, important final apoptotic enzymes involved with the downstream signaling of stress-activated protein kinase, were also activated after burn injury in muscle tissue from burned rats. These findings confirm the hypothesis that apoptosis occurs in skeletal muscle and that major apoptotic pathways are activated after a burn injury. Further characterization of these apoptotic signaling cascades may provide new therapeutic targets for the prevention of burn-induced muscle wasting.

Dose-related effects of single focal irradiation in the medial temporal lobe structures in rats--magnetic resonance imaging and histological study.

The dose-related effects of single focal irradiation on the medial temporal lobe in rats were investigated by sequential magnetic resonance imaging and histological examination. Irradiation of 200 Gy as a maximum dose using 4 mm collimators with a gamma unit created an area of necrosis consistently at the target site within 2 weeks after irradiation. Irradiation of 100 Gy caused necrosis within 10 weeks, and 75 Gy caused necrosis within one year. Irradiation of less than 50 Gy did not induce necrosis consistently, although a restricted area of necrosis was created in the medial temporal structures including the intraparenchymal portion of the optic tract. 75 Gy may be the optimum dose for creating necrosis consistently in the medial temporal lobe structures. However, careful dose planning considering both dose-time and dose-volume relationships in necrosis development is necessary to avoid injury to vulnerable neural structures such as the optic tract when applying radiosurgical techniques to treat functional brain disorders in medial temporal lobe structures such as temporal lobe epilepsy.

Functional role for protein kinase Cbeta as a regulator of stress-activated protein kinase activation and monocytic differentiation of myeloid leukemia cells.

Human myeloid leukemia cells respond to 12-O-tetradecanoylphorbol-13-acetate (TPA) and other activators of protein kinase C (PKC) with induction of monocytic differentiation. The present studies demonstrated that treatment of U-937 and HL-60 myeloid leukemia cells with TPA, phorbol-12,13-dibutyrate, or bryostatin 1 was associated with the induction of stress-activated protein kinase (SAPK). In contrast, TPA-resistant TUR and HL-525 cell variants deficient in PKCbeta failed to respond to activators of PKC with the induction of SAPK. A direct role for PKCbeta in TPA-induced SAPK activity in TUR and HL-525 cells that stably express PKCbeta was confirmed. We showed that TPA induced the association of PKCbeta with MEK kinase 1 (MEKK-1), an upstream effector of the SAPK/ERK kinase 1 (SEK1)-->SAPK cascade. The results also demonstrated that PKCbeta phosphorylated and activated MEKK-1 in vitro. The functional role of MEKK-1 in TPA-induced SAPK activity was further supported by the demonstration that the expression of a dominant negative MEKK-1 mutant abrogated this response. These findings indicate that PKCbeta activation is necessary for activation of the MEKK-1-->SEK1-->SAPK cascade in the TPA response of myeloid leukemia cells.

[Insulin receptor and aging].

The potential link between aging and insulin signaling has attracted substantial attention since several decades ago, on the basis of evidence including age-related increase in incidence of insulin resistance, insulin resistance and type 2 diabetes in accelerated aging syndromes and lifespan extension by caloric restriction in rodents. In addition, the intensive investigations in C. elegans in the 1990's, which have identified insulin signaling components including daf-2, age-1 and daf-16 as the genes whose mutations lead to lifespan extension, shed new light on molecular mechanisms underlying aging. As suggested by the genetic studies in C. elegans, it was recently demonstrated that FKHR, FKHRL1 and AFX, which are mammalian homologues of daf-16 forkhead transcription factor, function downstream of insulin signaling and Akt/PKB under cellular conditions. However, it is an open question whether insulin signaling components, including forkhead transcription factors, play a critical role in aging and longevity in mammals as well as in C. elegans. Increasing evidence concerning C. elegans indicates that augmented resistance to stress, in particular, that to oxidative stress is involved in lifespan extension by genetic mutations of insulin signaling components. The intriguing finding that signals from the reproductive system regulate lifespan by modulating the activities of insulin signal transduction pathway in C. elegans suggests a possibility of co-evolution of reproduction and aging. The significance of studies on C. elegans with regard to human aging is discussed.

Association of bone mineral density with apolipoprotein E phenotype.

The phenotypes of apolipoprotein E (Apo E) and their relationship with the bone mineral density (BMD) were examined in 284 unrelated postmenopausal Japanese women aged 47-82 years (64.0 +/- 1.0 years, mean +/- SE). The Apo E phenotype was analyzed by the isoelectric focusing method, followed by immunoblotting. The relationship between the Apo E phenotype and the vitamin D receptor (VDR) gene or estrogen receptor (ER) gene genotypes was also studied in the same population. The Apo E phenotypic frequencies in our population were 9.9% for E3/2, 66.5% for E3/3, 1.8% for E4/2, 19.7% for E4/3, and 2.1% for E4/4. We classified these phenotypes into three categories: Apo E4-/- (E3/2 and E3/3, n = 217, Apo E4 +/- (E4/3 and E4/2, n = 61), and Apo E4+/+ (E4/4, n = 6). The age, body weight, body height, and years since menopause were not significantly different among these three categories. The lumbar BMD values in these three groups were significantly different in the order of E4-/- (0.91 +/- 0.01 g/cm2), E4 +/- (0.85 +/- 0.02 g/cm2), and E4+/+ (0.83 +/- 0.06 g/cm2) (p = 0.031). The same trend was also observed for the Z score of the total BMD (p = 0.022). The serum level of intact osteocalcin in E4+/+ (15.2 +/- 5.7 ng/ml) was higher than in E4-/- (7.7 +/- 0.3 ng/ml) or E4 +/- (7.7 +/- 0.7 ng/ml) (p = 0.004 by analysis of variance). However, there were no other significant differences in the serum or urinary levels of bone turnover markers. Serum cholesterol in the E4+/+ group tended to be higher than in the other two groups (p = 0.05). There were no significant associations of the VDR and ER genotypes with the Apo E4 phenotype. A multivariate linear regression analysis revealed Apo E4 to be a significant, independent predictor of the Z score of the lumbar BMD. The effect of the Apo E4 allele on the Z score of the lumbar BMD (-0.493 +/- 0.152) was not significantly different from that in the AAB of VDR (-0.616 +/- 0.225) or PPxx of ER (-0.785 +/- 0.314). In conclusion, the Apo E4 allele is associated with a low bone mass in postmenopausal Japanese.

Activation of p38 mitogen-activated protein kinase by c-Abl-dependent and -independent mechanisms.

The p38 mitogen-activated protein (MAP) kinase defines a subgroup of the mammalian MAP kinases that are induced in response to lipopolysaccharide, hyperosmolarity, and interleukin 1. p38 MAP kinase appears to play a role in regulating inflammatory responses, including cytokine secretion and apoptosis. Here we show that diverse classes of DNA-damaging agents such as cisplatinum, 1-beta-D-arabinofuranosylcytosine, UV light, ionizing radiation, and methyl methanesulfonate activate p38 MAP kinase. We also demonstrate that cells deficient in c-Abl fail to activate p38 MAP kinase after treatment with cisplatinum and 1-beta-D-arabinofuranosylcytosine but not after exposure to UV and methyl methanesulfonate. Reconstitution of c-Abl in the Abl-/- cells restores that response. Similar results were obtained for induction of the Jun-NH2-kinase/stress-activated protein kinase. These findings indicate that p38 MAP and Jun-NH2-kinase/stress-activated protein kinases are differentially regulated in response to different classes of DNA-damaging agents.

Demonstration of isoforms of the estrogen receptor in the bone tissues and in osteoblastic cells.

Expression of isoforms of estrogen receptor (ER) was examined in the bone tissues. Reverse transcriptase-polymerase chain reaction ((RT-PCR) using specific primers for rat ER cDNA was performed with total RNA from rat bone tissues. Then, we sequenced the amplified products after cloning and identified two isoforms of the ER and the wild-type ER. One of the ER mRNA isoforms did not have the region corresponding to exon 4 and the other isoform did not have the region corresponding to both exon 3 and exon 4. These isoforms were designated as ER delta 4 isoform and ER delta 3/4 isoform, respectively. The existence of these isoforms was also confirmed by ROS-17/2.8 osteoblastic osteosarcoma cells. Chloramphenicol acetyltransferase assay showed that these isoforms lost estrogen dependent transactivation activities. We suggest that the ER isoforms may play some roles in the bone metabolism in which estrogen is essential to maintain bone density.

[Serum concentration of vitamin K in elderly women with involutional osteoporosis].

Oral administration of vitamin K was reported to increase bone mineral density. However, the possible role of vitamin K in the pathogenesis of osteoporosis still remains unclear. Therefore, we measured the serum concentration of vitamin K1 and K2 (menaquinone-4, 7, 8) in 24 elderly women with osteoporotic vertebral compression fracture and in 36 elderly women without fracture. Major forms of vitamin K present in sera in this study were vitamin K1 and menaquinone-7. On the other hand, serum menaquinone-4 and -8 were undetectable in most women. Serum concentration of menaquinone-7 was significantly lower in women with fracture than in those without fracture (3.29 +/- 3.63 ng/ml vs 6.26 +/- 5.62, mean +/- SD, respectively), while no difference was found in serum vitamin K1 concentration (0.837 +/- 0.620 ng/ml vs 0.820 +/- 0.686, respectively). There was no difference between both groups in background data such as age, body height, body weight, and body mass index, as well as serum level of calcium, inorganic phosphate, creatinine, albumin, and alkaline phosphatase. These results suggest the possibility that deficiency of vitamin K, particularly that of menaquinone-7, is one of the risk factors for developing osteoporosis.

Cell cycle-dependent expression of estrogen receptor and effect of estrogen on proliferation of synchronized human osteoblast-like osteosarcoma cells.

Dual fluoroimmunohistochemical staining of estrogen receptor (ER) and bromodeoxyuridine was performed in a human osteoblastic osteosarcoma cell line, HOS TE85 cells. ER immunoreactivity was observed preferentially in the nuclei of the cells that were bromodeoxyuridine positive. ER expression at various phases of the cell cycle was investigated in HOS TE85 cells, which were synchronized at the G1/S phase boundary by intermittent exposure to thymidine and hydroxyurea. ER immunoreactivity became detectable in the S phase, decreased in the G2/M and G1 phases, and then reappeared in the S phase of the next cell cycle. Western blot analysis also showed that ER protein exists in these cells and increases in the S phase. Moreover, Northern blot analysis demonstrated that the expression of ER messenger RNA increases in the early S phase, gradually decreases during the progress of the cell cycle, and increases again in the S phase of the subsequent cell cycle. Interestingly, 17 beta-estradiol (10(-8) M) increased cell number and [3H]thymidine incorporation into DNA in the synchronized HOS TE85 cells, whereas this effect was not observed in the nonsynchronized HOS TE85 cells. The present studies suggest that the cell cycle-dependent regulation may contribute to the heterogeneity of ER expression in osteoblastic cells.

Infarction of the unilateral posterior horn and lateral column of the spinal cord with sparing of posterior columns: demonstration by MRI.

Infarction of the spinal cord, particularly in the distribution of the posterior spinal arteries, is rare. Twenty seven cases of posterior spinal artery syndrome have been reported. In all cases, the posterior columns were affected, and in all but two, the lesions were bilateral. Here a 49-year-old woman is reported, who was affected with lumbar cord infarction in the distribution of the posterior spinal arteries, diagnosed by MRI and clinical presentation. This case is of special interest because of sparing of the posterior columns and the unilateral nature of the lesion, even though the infarction occurred in the distribution of the posterior spinal arteries.

Immunocytochemical identification of androgen receptor in mouse osteoclast-like multinucleated cells.

Expression of androgen receptor (AR) in mouse osteoclast-like multi-nucleated cells (OCs) was examined with immunocytochemical techniques. Murine OCs were obtained by co-culturing mouse osteoblastic cells and bone marrow cells. Three preparations of polyclonal anti-AR antibody which were raised in rabbit against different parts of the human AR were employed for the experiments. Specific staining for AR was demonstrated in the nuclei and the perinuclear area of mouse OCs. This is the first report demonstrating the presence of AR in osteoclast-like cells.

Effects of 1 alpha,25-dihydroxyvitamin D3 on macrophage colony-stimulating factor production and proliferation of human monocytic cells.

1 alpha-25-Dihydroxyvitamin D3 [1 alpha,25(OH)2D3] stimulates the proliferation of human monocytes in vitro. In the present study, we investigated a possible role of macrophage colony-stimulating factor (M-CSF) in 1 alpha,25(OH)2D3-induced proliferation of human circulating monocytes and the effects of 1 alpha,25(OH)2D3 on M-CSF production by human monocytic cells. Both 1 alpha,25(OH)2D3 and recombinant human M-CSF increased 2.5-fold the nucleus number of human circulating monocytes on day 6 of the culture. These effects were inhibited by antihuman M-CSF antibody as well as by anti-c-fms antibody, although these antibodies themselves did not affect the nucleus number when added to control culture. These results indicated that M-CSF is required for 1 alpha,25(OH)2D3-stimulated monocyte proliferation. In addition, 1 alpha,25(OH)2D3 stimulated M-CSF secretion from human circulating monocytes. Secretion and mRNA expression of M-CSF by 12-0-tetradecanoylphorbol-13-acetate (TPA)-treated THP-1 cells (human monocytic leukemia cell line) and TPA-treated HL-60 cells (human promyelocytic leukemia cell line) were also increased by 1 alpha,25(OH)2D3. M-CSF secretion from TPA-treated THP-1 cells was increased by 1 alpha,25(OH)2D3 in a dose-dependent and metabolite-specific manner. The present study demonstrates that 1 alpha,25(OH)2D3 is a potent stimulator for M-CSF production by human monocytic cells and that the proliferative effect of 1 alpha,25(OH)2D3 on human monocytes may be attributed, at least in part, to the stimulated secretion of M-CSF.