Cyclin-dependent Kinase Inhibitor Protein Research Articles

Haploinsufficiency of CDKN1B contributes to leukemogenesis in T-cell prolymphocytic leukemia

T-cell prolymphocytic leukemia (T-PLL) is consistently associated with inactivation of the ATM gene and chromosomal re-arrangements leading to an overexpression of MTCP1/TCL1 oncoproteins. These alterations are present at the earliest stage of malignant transformation, suggesting that additional events are required for overt malignancy. In this study, we pursued the investigation of the 12p13 deletion, previously shown to occur in approximately half of T-PLLs. We refined the minimal region of deletion by single nucleotide and microsatellite polymorphism allelotyping. We defined a 216-kb region containing the CDKN1B gene that encodes the cyclin-dependent kinase inhibitory protein p27(KIP1). Sequencing this gene in 47 T-PLL patient samples revealed a nonsense mutation in one case without 12p13 deletion. The absence of biallelic inactivation of CDKN1B for most patients suggested a haploinsufficiency mechanism for tumor suppression, which was investigated in an animal model of the disease. In a Cdkn1b(+/-) background, MTCP1 transgenics had consistent and multiple emergences of preleukemic clones not observed in control cohorts. The second Cdkn1b allele was maintained and expressed in these preleukemic clones. Altogether, these data strongly implicate CDKN1B haploinsufficiency in the pathogenesis of T-PLL.

Inhibition of Cell Cycle Progression by Hydroxytyrosol Is Associated with Upregulation of Cyclin-Dependent Protein Kinase Inhibitors p21WAF1/Cip1 and p27Kip1 and with Induction of Differentiation in HL60 Cells

Recent evidence indicates that the cancer preventive activity of olive oil can be mediated by the presence of minor components, such as antioxidant phenolic compounds. However, their mechanisms of action remain largely unknown. In this study, we investigated the in vitro effects of one of the main olive oil phenols, hydroxytyrosol [3,4-dihydroxyphenylethanol (3,4-DHPEA)], on proliferation, cell cycle progression, apoptosis, and differentiation of HL60 human promyelocytic leukemia cells. 3,4-DHPEA showed a potent inhibitory activity on DNA synthesis, as evidenced by a 92% reduction of [3H]-thymidine incorporation at 100 μmol/L, and an induced apoptosis, as evidenced by the release of cytosolic nucleosomes and flow cytometry. This phenol, 3,4-DHPEA, was also able to inhibit the progression of the cell cycle in synchronized HL60 cells, which accumulated in the G0/G1 phase of the cell cycle after 25 h of treatment. Furthermore, 3,4-DHPEA induced differentiation on HL60 cells with a maximum effect (22% of cells) at 100 μmol/L after 72 h of treatment. Among the different proteins involved in the regulation of the cell cycle, 3,4-DHPEA reduced the level of cyclin-dependent kinase (CDK) 6 and increased that of cyclin D3. With regard to the CDK inhibitors, p15 was not altered by 3,4-DHPEA treatment, whereas the expression of p21WAF1/Cip1 and p27Kip1 was increased at both protein and mRNA levels. To our knowledge, these results provide the first evidence that 3,4-DHPEA may effect the expression of genes involved in the regulation of tumor cell proliferation and differentiation.

Differential regulation of the cyclin-dependent kinase inhibitors p21 Cip1 and p27 Kip1 by phosphorylation directed by the cyclin encoded by Murine Herpesvirus 68

Members of the γ2-herpesvirus family encode cyclin-like proteins that have the ability to deregulate mammalian cell cycle control. Here we report the key features of the viral cyclin encoded by Murine Herpesvirus 68, M cyclin. M cyclin preferentially associated with and activated cdk2; the M cyclin/cdk2 holoenzyme displayed a strong reliance on phosphorylation of the cdk T loop for activity. cdk2 associated with M cyclin exhibited substantial resistance to the cdk inhibitor proteins p21 Cip and p27 Kip. Furthermore, M cyclin directed cdk2 to phosphorylate p27 Kip1 on threonine 187 (T187) and cellular expression of M cyclin led to down-regulation of p27 Kip1 and the partial subversion of the associated G1 arrest. Mutation of T187 to a non-phosphorylatable alanine rendered the p27 Kip1-imposed G1 arrest resistant to M cyclin expression. Unlike the related K cyclin, M cyclin was unable to circumvent the G1 arrest associated with p21 Cip1 and was unable to direct its associated catalytic subunit to phosphorylate this cdk inhibitor. These results imply that M cyclin has properties that are distinct from other viral cyclins and that M cyclin expression alone is insufficient for S phase entry.

Sonic Hedgehog Stimulates Mouse Embryonic Stem Cell Proliferation by Cooperation of Ca2+/Protein Kinase C and Epidermal Growth Factor Receptor As Well as Gli1 Activation

Hedgehog signaling has an essential role in the control of stem cell growth in embryonic tissues. Therefore, this study examined the effect of sonic hedgehog (Shh) on the self-renewal of mouse embryonic stem (ES) cells and its related mechanisms. Shh increased DNA synthesis blocked by the inhibition of the smoothened receptor. Shh required Gli1 activation to induce the increases in Notch/Hes-1 and Wnt/beta-catenin. Shh increased the intracellular calcium concentration ([Ca(2+)](i)) and protein kinase C (PKC) activity. We show that the Shh-induced increase in the Gli1 mRNA level requires [Ca(2+)](i) and PKC. Shh increased the phosphorylation of epidermal growth factor receptor (EGFR), which is blocked by the matrix metalloproteinase inhibitor. Subsequently, Shh increased the nuclear factor (NF)-kappaB p65 phosphorylation, which was inhibited by blocking PKC and EGFR tyrosine kinase. Shh also increased the level of the cell cycle regulatory proteins in a dose-dependent manner. However, Shh decreased the levels of the cyclin-dependent kinase inhibitory proteins. The effect of Shh on these proteins was inhibited by blocking PKC, EGFR, and NF-kappaB as well as transfection of Gli1 small interfering RNA (siRNA). Finally, Shh-induced progression of the G1/S-phase was blocked by the inhibition of PKC and EGFR tyrosine kinase. In conclusion, Shh stimulates mouse ES cell proliferation through Gli1 activation as well as Ca(2+)/PKC and EGFR. Disclosure of potential conflicts of interest is found at the end of this article.

Strain related changes in regional myocardial cyclin-dependent kinase (Cdk) inhibitor protein, p21 post-myocardial infarction in ovine model

Yankey, Godfred K. MD; Li, Teiluo MD; Kilic, Ahmet MD; Savai, Kinjal MS; Cheng, Guangming MD; Satpute, Aditee MD; Prastein, Deyanira MD; Wu, Zhongjun PhD; Griffith, Bartley MD Author Information

Acteoside inhibits human promyelocytic HL-60 leukemia cell proliferation via inducing cell cycle arrest at G0/G1 phase and differentiation into monocyte

We investigated the in vitro effects of acteoside on the proliferation, cell cycle regulation and differentiation of HL-60 human promyelocytic leukemia cells. Acteoside inhibited the proliferation of HL-60 cells in a concentration- and time-dependent manner with an IC50, approximately 30 microM. DNA flow cytometric analysis indicated that acteoside blocked cell cycle progression at the G1 phase in HL-60 human promyelocytic leukemia cells. Among the G1 phase cell cycle-related proteins, the levels of cyclin-dependent protein kinase (CDK)2, CDK6, cyclin D1, cyclin D2, cyclin D3 and cyclin E were reduced by acteoside, whereas the steady-state level of CDK4 was unaffected. The protein and mRNA levels of CDK inhibitors (cyclin-dependent kinase inhibitors), such as p21(CIP1/WAF1) and p27(KIP1), were gradually increased after acteoside treatment in a time-dependent manner. In addition, acteoside markedly enhanced the binding of p21(CIP1/WAF1) and p27(KIP1) to CDK4 and CDK6, resulting in the reduction of CDK2, CDK4 and CDK6 activities. Moreover, the hypophosphorylated form of retinoblastoma increased, leading to the enhanced binding of protein retinoblastoma (pRb) and E2F1. Our results further suggest that acteoside is a potent inducer of differentiation of HL-60 cells based on biochemical activities and the expression level of CD14 cell surface antigen. In conclusion, the onset of acteoside-induced G1 arrest of HL-60 cells prior to the differentiation appears to be tightly linked to up-regulation of the p21(CIP1/WAF1) and p27(KIP1) levels and decreases in the CDK2, CDK4 and CDK6 activities. These findings, for the first time, reveal the mechanism underlying the anti-proliferative effect of acteoside on human promyelocytic HL-60 cells.

Effects of cyclin dependent protein kinase inhibitor olomoucine on the neuronal apoptosis after status epilepticus: experiment with rats

To investigate the effects of olomoucine, a cyclin dependent protein kinase (CDK) inhibitor, on the neuronal apoptosis after status epilepticus (SE). Lithium chloride was injected intraperitoneally, and pilocarpine was injected intraperitoneally after 18 h to 24 SD rats so as to cause SE. Twenty-two of the 24 rats developed SE and 2 of them died. The surviving 20 rats were then randomly divided into 2 equal groups: olomoucine group, injected intracerebroventricularly after the SE was terminated by diazepam and chloral hydrate once a day for 3 days, and SE group, infused intracerebroventricularly with DMSO solution Another 10 rats were injected intraperitoneally with normal saline and then infused intracerebroventricularly with DMSO solution to be used as control group. Six hours after SE attack 5 rats from each group were killed respectively with their brains taken out. Semiquantitative RT-PCR was used to detect the mRNA expression of anti-inflammatory cytokines, such as interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. Three days later the other 5 rats in each group were killed with their entorhinal cortex and hippocampus taken out. TUNEL was used to observe the apoptosis. Immunofluorescence (IF) staining was used to detect the expression of neuronal nuclear nucleoprotein (NeuN) and cyclin B1. TUNEL showed that apoptotic neurons were rare in the control group and were numerous in the SE group, especially in the entorhinal cortex and the hylus of dentate gyrus, and the number of apoptotic neurons in the hylus of dentate gyrus of the olomoucine group was not significantly different from that of the control group (P < 0.05), however, the number of apoptotic cells in the entorhinal cortex of the olomoucine group was still significantly higher than that of the control group (P < 0.05). IF staining demonstrated that in the control group the co-expression of NeuN and TUNEL-labeled cells was weak; and in the SE group the co-expression of NeuN and TUNEL was significantly increased compared with that in the control group (P < 0. 05). The number of cyclin B1 positive cells in the olomoucine group was 18.22 +/- 3.99, significantly lower than that of the SE group (24.57 +/- 6.78, P < 0.05). Semiquantitative RT-PCR showed that the IL-1beta and TNF-alpha mRNA expression levels of the SE group were both significantly higher than those of the control group (both P < 0.05), and the IL-1beta and TNF-alpha mRNA expression levels of the olomoucine group, except the TNF-alpha mRNA expression in the cortex, were all significantly lower than those of the SE group (all P < 0.05), and not significantly different from those of the control group (all P > 0.05). Olomoucine treatment can inhibits cell cycle protein B1 expression, anti-inflammatory cytokines such as IL-1beta and TNF-alpha secretion, thus decreasing neuronal death and providing neuroprotection after SE, which suggests a potential promising therapeutic way for epilepsy treatment.

Reduced XPC messenger RNA level may predict a poor outcome of patients with nonsmall cell lung cancer

Homologous deletion of the xeroderma pigmentosum complementary group C (XPC) repair gene frequently causes lung adenocarcinoma in mice, suggesting that an XPC defect may play a critical role in lung tumorigenesis. The current study attempted to determine whether reduced XPC mRNA levels predict the clinical outcome of lung cancer patients. XPC, p27(kip) (cdk inhibitory protein), and S-phase kinase-associated protein (skp2) levels were evaluated by Western blot analysis in a series of lung cancer cell lines with different invasive abilities. Migration and invasive abilities were measured using a modified Boyden chamber without and with Matrigel, respectively. To test whether XPC affects cell invasive ability, XPC gene protein expression was reduced in low invasive cells by RNA interference (RNAi) and assayed with Boyden chamber. XPC mRNA levels in 126 nonsmall cell lung cancers (NSCLCs) were examined by real-time-reverse-transcriptase polymerase chain reaction (RT-PCR). The prognostic value of XPC mRNA expression was statistically analyzed by the Kaplan-Meier method and Cox proportional hazards regression. The expression of XPC was reduced with increasing invasive potential in CL1-series lung cancer cell lines. When the XPC level was reduced by RNAi, cell migration and invasiveness increased markedly; the increased invasiveness may be caused by decreased expression of p27(kip) and increased expression of skp2 and E2F transcription factor 1. To determine whether reduced XPC expression was correlated with tumor aggressiveness and poor patient survival, XPC mRNA levels were evaluated by real-time RT-PCR. Kaplan-Meier analysis demonstrated that the median survival of patients with lower XPC mRNA levels was shorter compared with patients with higher XPC mRNA levels (P = .0440). Cox regression analysis further indicated that XPC mRNA level may act as an independent prognostic factor for NSCLC patients (P = .014). The results of the current study suggest that reduced XPC mRNA level may constitute an independent prognostic factor for NSCLC patients.

Antiproliferative Autoantigen CDA1 Transcriptionally Up-regulates p21Waf1/Cip1 by Activating p53 and MEK/ERK1/2 MAPK Pathways

We previously reported that overexpression of cell division autoantigen 1 (CDA1) in HeLa cells arrests cell growth and inhibits DNA synthesis at S-phase. Here we show that CDA1-induced arrest of cell growth is accompanied by increases in protein and mRNA levels of the cyclin-dependent kinase (Cdk) inhibitor protein, p21(Waf1/Cip1) (p21). Both p21 induction and cell growth arrest are reversed when CDA1 expression is inhibited. CDA1 also increases p53 protein, but not its mRNA, in a time- and dose-dependent manner. MDM2, a ubiquitin ligase regulating p53 degradation, is inactivated by CDA1, suggesting that p53 protein accumulation is due to decreased protein degradation. Knockdown of p53, using siRNA targeting two sites of p53 mRNA, abrogates transcriptional induction of p21 by CDA1. Deletion of the p53 responsive element in the distal region of p21 promoter attenuates promoter activity in response to CDA1. DNA damage caused by camptothecin treatment increases mRNA and protein levels of CDA1, accompanied by induction of p53. The DNA damage-induced p53 induction is markedly attenuated by CDA1 knockdown. CDA1 induces phosphorylation of ERK1/2(p44/42), an activity blocked by PD98059 and U0126, inhibitors of the upstream kinase MEK1/2. The MEK inhibitors also block induction of p21 mRNA and abrogate p21 promoter activity stimulated by CDA1. Cell cycle kinases, Cdk1, -2, -4, and -6 are inhibited by CDA1 overexpression. We conclude that CDA1 induces p53- and MEK/ERK1/2 MAPK-dependent expression of p21 by acting through the p53 responsive element in the p21 promoter and that this contributes to its antiproliferative activity.

Gold Nanoparticles Inhibit the Proliferation of Multiple Myeloma Cells

Gold nanoparticles significantly inhibit proliferation in multiple myeloma cell lines (see figure) in a dose-dependent manner. Gold-treated multiple myeloma cells show cell-cycle arrest in the G1-phase (the phase when chromosomes prepare for replication) by up-regulation of cell-cycle cyclin-dependent kinase inhibitor proteins p21 and p27. Gold nanoparticles do not affect normal peripheral blood mononuclear cells.

A Mechanism for Cell-Cycle Regulation of MAP Kinase Signaling in a Yeast Differentiation Pathway

Yeast cells arrest in the G1 phase of the cell cycle upon exposure to mating pheromones. As cells commit to a new cycle, G1 CDK activity (Cln/CDK) inhibits signaling through the mating MAPK cascade. Here we show that the target of this inhibition is Ste5, the MAPK cascade scaffold protein. Cln/CDK disrupts Ste5 membrane localization by phosphorylating a cluster of sites that flank a small, basic, membrane-binding motif in Ste5. Effective inhibition of Ste5 signaling requires multiple phosphorylation sites and a substantial accumulation of negative charge, which suggests that Ste5 acts as a sensor for high G1 CDK activity. Thus, Ste5 is an integration point for both external and internal signals. When Ste5 cannot be phosphorylated, pheromone triggers an aberrant arrest of cells outside G1 either in the presence or absence of the CDK-inhibitor protein Far1. These findings define a mechanism and physiological benefit of restricting antiproliferative signaling to G1.

Rapamycin‐mediated G1 arrest involves regulation of the Cdk inhibitor Sic1 in Saccharomyces cerevisiae

The rapamycin-sensitive (TOR) signalling pathway in Saccharomyces cerevisiae controls growth and cell proliferation in response to nutrient availability. Rapamycin treatment causes cells to arrest growth in G1 phase. The mechanism by which the inhibition of the TOR pathway regulates cell cycle progression is not completely understood. Here we show that rapamycin causes G1 arrest by a dual mechanism that comprises downregulation of the G1-cyclins Cln1-3 and upregulation of the Cdk inhibitor protein Sic1. The increase of Sic1 level is mostly independent of the downregulation of the G1 cyclins, being unaffected by ectopic CLN2 expression, but requires Sic1 phosphorylation of Thr173, because it is lost in cells expressing Sic1(T173A). Rapamycin-mediated Sic1 upregulation involves nuclear accumulation of a more stable, non-ubiquitinated protein. Either SIC1 deletion or CLN3 overexpression results in non-cell-cycle-specific arrest upon rapamycin treatment and makes cells sensitive to a sublethal dose of rapamycin and to nutrient starvation. In conclusion, our data indicate that Sic1 is involved in rapamycin-induced G1 arrest and that deregulated entrance into S phase severely decreases the ability of a cell to cope with starvation conditions induced by nutrient depletion or which are mimicked by rapamycin treatment.

ANG II‐stimulated DNA synthesis is mediated by ANG II receptor‐dependent Ca2+/PKC as well as EGF receptor‐dependent PI3K/Akt/mTOR/p70S6K1 signal pathways in mouse embryonic stem cells

Effect of angiotensin II (ANG II) on mouse embryonic stem (ES) cell proliferation was examined. ANG II increased [(3)H] thymidine incorporation in a time- (>4 h) and dose- (>10(-9) M) dependent manner. The ANG II-induced increase in [(3)H] thymidine incorporation was blocked by inhibition of ANG II type 1 (AT(1)) receptor but not by ANG II type 2 (AT(2)) receptor, and AT(1) receptor was expressed. ANG II increased inositol phosphates formation and [Ca(2+)](i), and translocated PKC alpha, delta, and zeta to the membrane fraction. Consequently, the inhibition of PLC/PKC suppressed ANG II-induced increase in [(3)H] thymidine incorporation. The inhibition of EGF receptor kinase or tyrosine kinase prevented ANG II-induced increase in [(3)H] thymidine incorporation. ANG II phosphorylated EGF receptor and increased Akt, mTOR, and p70S6K1 phosphorylation blocked by AG 1478 (EGF receptor kinase blocker). ANG II-induced increase in [(3)H] thymidine incorporation was blocked by the inhibition of p44/42 MAPKs but not by p38 MAPK inhibition. Indeed, ANG II phosphorylated p44/42 MAPKs, which was prevented by the inhibition of the PKC and AT(1) receptor. ANG II increased c-fos, c-jun, and c-myc levels. ANG II also increased the protein levels of cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK4 but decreased the p21(cip1/waf1) and p27(kip1), CDK inhibitory proteins. These proteins were blocked by the inhibition of AT(1) receptor, PLC/PKC, p44/42 MAPKs, EGF receptor, or tyrosine kinase. In conclusion, ANG II-stimulated DNA synthesis is mediated by ANG II receptor-dependent Ca(2+)/PKC and EGF receptor-dependent PI3K/Akt/mTOR/p70S6K1 signal pathways in mouse ES cells.

HiNF‐P is a bifunctional regulator of cell cycle controlled histone H4 gene transcription

Cell cycle progression beyond the G1/S phase transition requires the activation of a transcription complex containing histone nuclear factor P (HiNF-P) and nuclear protein mapped to ataxia telangiectasia (p220(NPAT)) in response to cyclin dependent kinase 2 (CDK2)/cyclin E signaling. We show here that the potent co-activating properties of HiNF-P/p220(NPAT) on the histone H4 gene promoter, which are evident in the majority of human cell types, are sporadically neutralized in distinct somatic cell lines. In cells where HiNF-P and p220(NPAT) do not activate the H4 gene promoter, HiNF-P instead represses transcription. Our data suggest that the cell type specific expression of the cyclin-dependent kinase inhibitory (CKI) protein p57(KIP2) inhibits the HiNF-P dependent activation of the histone H4 promoter. We propose that, analogous to E2F proteins and other cell cycle regulatory proteins, HiNF-P is a bifunctional transcriptional regulator that can activate or repress cell cycle controlled genes depending on the cellular context.

HepG2 인체간암세포의 세포주기조절인자 발현에 미치는 sulforaphane의 영향

브로콜리를 포함한 십자화과 식물에서 glucoraphanin의 가수분해를 통해 생성되는 isothiocyanate의 일종인 sulforaphane은 역학적 조사를 포함한 다양한 선행 연구에서 강력한 암예방 효과를 가지는 것으로 알려져 있다. 항암효과에 관한 최근 연구 결과에 따르면 sulforaphane은 다양한 인체암세포의 증식을 억제하고 apoptosis를 유발할 수 있는 것으로 알려지고 있으나, 정확한 분자생물학적 기전은 밝혀져 있지 않은 상태이다. 본 연구에서는 sulforaphane의 항암작용 기전을 조사하기 위하여 HepG2 인체간암세포의 증식에 미치는 sulforaphane의 영 향을 조사하였다. Sulforaphane의 처리에 의한 HepG2 세포의 증식억제 및 형태적 변형은 세포주기 G2/M arrest 및 apoptosis 유발과 밀접한 관련이 있음을 알 수 있었다. RT-PCR 및 Western blot 분석 결과, sulforaphane 처리에 의하여 cyclin A 및 cyclin B1, Cdc2의 발현이 단백질 수준에서 선택적으로 저하되었으며, 종양억제 유전자 p53 및 Cdk inhibitor p21의 발현은 전사 및 번역 수준에서 sulforaphane 처리 농도 의존적으로 증가되었다. Sulforaphane의 항암 기전을 규명하기 위해서는 더 많은 연구가 부가적으로 필요하겠지만, 본 연구의 결과들에 의하면 sulforaphane은 강력한 인체암세포의 증식 억제 및 항암작용이 있을 것을 시사하여 준다고 할 수 있다. Sulforaphane, an isothiocyanate derived from hydrolysis of glucoraphanin in broccoli and other cruciferous vegetables, was shown to induce phase II detoxification enzymes and inhibit chemically induced mammary tumors in rodents. Recently, sulforaphane is known to induce cell cycle arrest and apoptosis in human canter cells, however its molecular mechanisms are poorly understood. In tile present study, we demonstrated that sulforaphane acted to inhibit proliferation and induce morphological changes of human hepatocarcinoma HepG2 cells. Treatment of HepG2 cells with <TEX>$10{\mu}M\;or\;15{\mu}M$</TEX> sulforaphane resulted in significant G2/M cell cycle arrest as determined by DNA flow cytometry. Moreover, <TEX>$20{\mu}M$</TEX> sulforaphane significantly induced the population of sub-G1 cells suggesting that sulforaphane induced apoptosis. This anti-proliferative effect of sulforaphane was accompanied by a marked inhibition of ryclin A, cyclin 31 and Cdc2 protein. However, the levels of tumor suppressor p53 and Cdk inhibitor p21 mRNA and protein expression were significantly increased by sulforaphane treatment in a concentration-dependent manner. Although further studies are needed, the present work suggests that sulforaphane may be a potential rhemoprevetiveichemotherapeucc agent for the treatment of human cancer cells.

Thioredoxin overexpression in mice, model of attenuation of oxidative stress, prevents benzene-induced hemato-lymphoid toxicity and thymic lymphoma

Reactive oxygen species (ROS), generated following benzene exposure, are considered to trigger the development of hematopoietic neoplasms, although little supporting evidence has been found. In this study, we examined whether the experimental elimination of ROS generated following benzene exposure prevents the development of benzene-induced hematopoietic disorders to clarify the mechanism underlying the development of benzene-induced hematopoietic disorders. C57BL/6 mice, overexpressing human thioredoxin (h-Trx-Tg), were used to examine the possible nullification of ROS induction following benzene exposure. The experimental group was exposed to 300 ppm benzene 6 hours/day, 5 days/week, for 26 weeks, and lifetime observation followed by molecular and histopathological examinations were carried out. The present study using h-Trx-Tg mice showed a complete suppression of the development of thymic lymphoma induced by benzene inhalation (0% in h-Trx-Tg vs 30% in wild-type (Wt) mice). This was associated with a 48% decrease in the incidence of clastogenic micronucleated reticulocyte induction in the h-Trx-Tg mice compared with the Wt control after 2 weeks of inhalation. As underlying mechanisms, the attenuation of oxidative stress was accompanied by a complete abrogation of hemato-lymphoid toxicity, as shown by the upregulation of the activity of superoxide-dismutase, and a consequently stable ROS level, as determined by cell sorting using 2', 7'-dichlorodihydrofluorescein diacetate, along with a significant attenuation of the overexpression of a cell cycle-dependent kinase inhibitor, p21. The attenuation of benzene-induced oxidative stress and that of the consequent lymphomagenesis were observed for the first time, and these indicate a role of oxidative stress in benzene-induced clastogenesis and lymphomagenesis. (These attenuations were not seen in nonthymic lymphomas, and no leukemias developed in C57BL/6 used in this study.) During the constitutive overexpression of h-Trx, the expression of aryl-hydrocarbon receptor in h-Trx-Tg mice was downregulated, which may also contribute to the attenuation.

Cyclin D1 Induction of Cellular Migration Requires p27KIP1

The cyclin D1 gene is amplified and overexpressed in human breast cancer, functioning as a collaborative oncogene. As the regulatory subunit of a holoenzyme phosphorylating Rb, cyclin D1 promotes cell cycle progression and a noncatalytic function has been described to sequester the cyclin-dependent kinase inhibitor protein p27. Cyclin D1 overexpression correlates with tumor metastasis and cyclin D1-deficient fibroblasts are defective in migration. The genetic mechanism by which cyclin D1 promotes migration and movement is poorly understood. Herein, cyclin D1 promoted cellular migration and cytokinesis of mammary epithelial cells. Cyclin D1 enhanced cellular migratory velocity. The induction of migration by cyclin D1 was abolished by mutation of K112 or deletion of NH(2)-terminal residues 46 to 90. These mutations of cyclin D1 abrogated physical interaction with p27(KIP1). Cyclin D1(-/-) cells were p27(KIP1) deficient and the defect in migration was rescued by p27(KIP1) reintroduction. Conversely, the cyclin D1 rescue of cyclin D1(-/-) cellular migration was reversed by p27(KIP1) small interfering RNA. Cyclin D1 regulated p27(KIP1) abundance at the posttranslational level, inhibiting the Skp2 promoter, Skp2 abundance, and induced p27(KIP1) phosphorylation at Ser(10). Together, these studies show cyclin D1 promotes mammary epithelial cell migration. p27(KIP1) is required for cyclin D1-mediated cellular migration.

Melatonin‐induced gene expression changes and its preventive effects on adriamycin‐induced lipid peroxidation in rat liver

Adriamycin (ADR) provokes lipid peroxidation process, while melatonin (MEL) is a free radical scavenger that has been found to protect against lipid peroxidation in vitro and in many experimental models. In the present study, the effects of ADR and the combination of ADR and MEL were analyzed on the modulation of fatty acid composition, lipid peroxidation and gene expression in rat liver. Sixty genes were selected for the study of relative gene expression changes in the liver. ADR treatment decreased the polyunsaturated fatty acids C22:6 n-3 and C20:4 n-6 in rat liver mitochondria. When the treatment of ADR was followed by MEL, decrease in these fatty acids could not be detected. A significant increase in lipid peroxidation was observed after administration of ADR, which was restored to control values by post-treatment with MEL. Gene expression profiles of ADR- versus ADR+MEL-treated rat livers indicated that both treatments induced significant changes. Quantitative real-time polymerase chain reaction analysis of 60 genes involved in oxidative stress revealed that cyp1b1, which is involved in electron transport, cyclin-dependent kinase inhibitor 1a that possesses cyclin-dependent protein kinase inhibitor activity, was induced at a more pronounced level in the ADR+MEL-treated samples than in the ADR-treated ones. Several genes having roles in heat-shock response were downregulated in MEL-treated animals, such as hsp40, hsp70 and hsp90 proteins reflecting the reduced oxidative stress in these animals. Global gene expression analysis will highlight the gene expression changes accompanying oxidative damage and its prevention in more details.

Oncostatin M Induces Growth Arrest by Inhibition of Skp2, Cks1, and Cyclin A Expression and Induced p21 Expression

Oncostatin M has been characterized as a potent growth inhibitor for various tumor cells. Oncostatin M-treated glioblastoma cells cease proliferation and instigate astrocytal differentiation. The oncostatin M-induced cell cycle arrest in G(1) phase is characterized by increased level of the cyclin-dependent kinase (CDK) inhibitory proteins p21(Cip1/Waf1/Sdi1) and p27(Kip1). Induction of p21 protein corresponds to increased mRNA level, whereas p27 accumulates due to increased stability of the protein. Interestingly, stabilization of p27(Kip1) occurs even in S phase, showing that p27 stabilization is a direct consequence of oncostatin M signaling and not a result of the cell cycle arrest. Degradation of p27 in late G(1) and S phase is initiated by the ubiquitin ligase complex SCF-Skp2/Cks1. Oncostatin M inhibits expression of two components of this E3 ligase complex (Skp2 and Cks1). Although combined overexpression of Skp2 and Cks1 rescues p27 degradation in S phase, it can not override p27 accumulation in G(1) phase and cell cycle arrest by oncostatin M. In addition to increasing Cdk inhibitor level, oncostatin M also impairs cyclin A expression. Cyclin A mRNA and protein level decline shortly after oncostatin M addition. The accumulation of two CDK inhibitor proteins and the repression of cyclin A expression may explain the broad and potent antiproliferative effect of the cytokine.

Effects of cyclin dependent protein kinase inhibitor olomoucine on the microenvironment of axonal regeneration after spinal cord injury: an experiment with rats

To investigate the effects of olomoucine, a cyclin dependent protein kinase (CDK) inhibitor, on the microenvironment of axonal regeneration after spinal cord injury (SCI). Forty-five SD rats were randomly divided into 3 equal groups: SCI group undergoing SCI by hemisection technique and peritoneal injection of dimethyl sulfoxide (DMSO) solution 30 min after the SCI, SCI + olomoucine (SCI + Olo) group undergoing SCI by hemisection technique and peritoneal injection of olomoucine solution 30 min after the SCI, and sham operation group undergoing sham operation and peritoneal injection of DMSO solution 30 min after the operation. Three days after the operation the injured spinal cord segments of 5 rats from each group were taken out. Western blotting was used to detect the expression of the cell cycle related proteins, cyclin A, cyclin B, cyclin E, and proliferating cell nuclear antigen (PCNA). Immunofluorescence (IF) staining was used to detect the expression of glial fibrillary acidic protein (GFAP), growth associated protein-43 (GAP-43) and chondroitin sulphate proteoglycan (CSPG). Four weeks after the operation specimens of the injured spinal cord segment 15 mm in length were taken out from 5 rats in each group to undergo histological examination. The locomotion function of the hindlimbs was determined by modified Gale combined behavioral scoring (SBS) 1 day and 1, 2, 4, 6, and 8 weeks after the operation. Western blotting 3 days after the operation showed that the expressions of cyclin A, cyclin B, cyclin E, and PCNA were very weak in the sham operation group, were significantly increased in the SCI group, and were significantly down-regulated in the SCI + Olo group compared with those of the SCI group. IF staining showed that the number of astrocytes was small and the expressions of GFAP, CSPG, and GAP-43 were weak in the sham operation group; in the SCI group the astrocytic proliferation and glial scar was obvious, and the expressions of GFAP, CSPG, and GAP-43 were significantly increased compared with those of the sham operation group (all P < 0.05); and the astrocytic proliferation was significantly weaker and no obvious glial scar could be seen, and the expressions of GFAP and CSPG were weaker in the SCI + Olo group in comparison with the SCI group, however, the GAP-43 expression of the sham operation group was significantly increased compared with that of the sham operation group (P < 0.05). The hindlimbs of the SCI + Olo group and sham operation group were paralyzed without significant difference in the CBS values between these 2 groups, however, two weeks after the operation, the locomotion function scores at different time points of the SCI + Olo group were all significantly improved in comparison with that of the SCI group (all P < 0.05). Olomoucine promotes the recovery of the locomotion function of the paralyzed hindlimbs, probably through microenvironmental improvement of axonal regeneration by inhibiting the glial scar formation and CSPG secretion as well as upregulating the GAP-43 expression.