Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

AMPK activators suppress cervical cancer cell growth through inhibition of DVL3 mediated Wnt/β-catenin signaling activity.

Recent evidence has suggested that AMPK activators may be applied as therapeutic drugs in suppressing cancer cell growth. However, the molecular mechanism of their suppressive function in cancer cells is still unclear. Here we show that AMPK activators impair cervical cancer cell growth through the reduction of DVL3, a positive regulator in Wnt/β-catenin signaling and an oncogenic player in cervical cancer tumorigenesis. By western blot and immunohistochemical analyses, we demonstrated that DVL3 was frequently upregulated and significantly associated with elevated β-catenin (P = 0.009) and CyclinD1 (P = 0.009) expressions in cervical cancer. Enforced expression of DVL3 elevated β-catenin and augmented cervical cancer cell growth, verifying that DVL3-mediated Wnt/β-catenin activation is involved in cervical cancer oncogenesis. On the other aspect, we noted that the cervical cancer cell growth was remarkably suppressed by AMPK activators and such cell growth inhibition was in concomitant with the reduction of DVL3 protein level in dose- and time-dependent manners. Besides, impaired mTOR signaling activity also reduced DVL3 expression. In contrast, co-treatment with Compound C (AMPK inhibitor) could significantly abrogate metformin induced DVL3 reduction. In addition, co-treatment with AM114 or MG132 (proteosomal inhibitors) could partially restore DVL3 expression under the treatment of metformin. Further in vivo ubiquitination assay revealed that metformin could reduce DVL3 by ubiquitin/proteasomal degradation. To our knowledge, this is the first report showing the probable molecular mechanisms of that the AMPK activators suppress cervical cancer cell growth by impairing DVL3 protein synthesis via AMPK/mTOR signaling and/or partially promoting the proteasomal degradation of DVL3

Targeting GRB7/ERK/FOXM1 Signaling Pathway Impairs Aggressiveness of Ovarian Cancer Cells

<div><p>Ovarian cancer is a highly lethal disease with poor prognosis and especially in high-grade tumor. Emerging evidence has reported that aberrant upregulation and activation of GRB7, ERK as well as FOXM1 are closely associated with aggresivenesss of human cancers. However, the interplay between these factors in the pathogenesis of human cancers still remains unclear. In this study, we found that GRB7 (<em>P</em><0.0001), ERK phosphorylation (<em>P</em><0.0001) and FOXM1 (<em>P</em> = 0.001) were frequently increased and associated with high-grade tumors, as well as a high tendency in association with advanced stage ovarian cancer by immunohistochemical analysis. Intriguingly, the expressions of GRB7 (<em>P</em><0.0001), ERK phosphorylation (<em>P</em><0.001) and FOXM1 (<em>P</em><0.001) showed a significant stepwise increase pattern along Grade 1 to Grade 3 ovarian cancers. Biochemical studies using western blot analysis demonstrated that enforced expression or knockdown of GRB7 showed GRB7 could elevate the levels of ERK phosphorylation and FOXM1, whereas enforced expression of FOXM1 could not alter levels of GRB7 and ERK phosphorylation. But inhibition of ERK signaling by U0126 or PD98059 could reduce the level of FOXM1 in GRB7-overexpressing ovarian cancer cells, suggesting that GRB7, ERK and FOXM1 are regulated orderly. Moreover, inhibition of ERK activity by U0126 or PD98059, or decreased FOXM1 expression by Thiostrepton significantly inhibited cell migration/invasion, tumor growth <em>in vitro</em> and <em>in vivo</em>. Collectively, our findings confer that targeting GRB7/ERK/FOXM1 signaling cascade may be a promising molecular therapeutic choice in combating ovarian cancer.</p> </div

DVL3 and β-catenin are frequently up-regulated and associated with augmented Wnt/β-catenin signaling activity in cervical cancer.

<p>(A) Western blot analysis showed that only the level of DVL3 but not DVL1 and DVL2 was significantly up-regulated in cervical cancer cell lines when compared with the normal cervix cell lines. (B) Real time q-PCR revealed the mRNA of <i>DVL3</i> was obviously higher among cervical cancer cell lines when compared with the normal cervix cell lines. The value of NC105 was used to normalize other cell lines. (C) Immunohistochemical analysis showed that both DVL3 and β-catenin were upregulated on cervical cancer tissues when compared with normal cervix samples. DVL3 was distributed in the cytoplasmic region whereas β-catenin was located in both the cytoplasmic and nuclear regions. (Magnification: 20x) (D) Luciferase reporter assay revealed that transient transfection of DVL3-expressing plasmid could increase β-catenin transcriptional activity in HEK293 cells.</p

Clinicopathological correlation of β-catenin expression in cervical cancer tissue array (CX1021) (Pantomics, CA, USA).

<p>Clinicopathological correlation of β-catenin expression in cervical cancer tissue array (CX1021) (Pantomics, CA, USA).</p

AMPK activation impairs cervical cancer cell growth through the reduction of DVL3.

<p>(A) Real time q-PCR analysis demonstrated that metformin had no effect on the expression of <i>DVL3</i> mRNA expression in C33A and HeLa cells. (B) Western blot analysis revealed that compound C could abate the decrement of DVL3 mediated by metformin. (C) XTT cell proliferation assay showed a significant reduction of cell proliferation in C33A (<i>P</i>>0.001), SiHa (<i>P</i>>0.001) and HeLa cells (<i>P</i>>0.001) treated with metformin in a dose-dependent manner. (D) Clonogenic assay showed a remarkable reduction of the number of colonies formed in HeLa and C33A treated with metformin in a dose-dependent manner.</p

AMPK activators reduce DVL3 in cervical cancer cells.

<p>(A) Upon treatment of AICAR, approximately 80% reduction of DVL3 level was observed among C33A, C41 and CaSki. (B) Upon treatment of A769662 at different doses (0, 50 and 100 μM), a 20-30% and 80%–100% reduction of DVL3 in SiHa and C33A could be observed respectively. (C) Upon treatment with A23187 at different doses (0, 1.25 and 2.5 μM) for twenty hours, C33A and SiHa cells showed 80–90% reduction of DVL3 expression in a dose-dependent manner. (D) Upon treatment of 1.25 μM A23187, the decrease of DVL3 could be observed after 10 and 12 hours in C33A and CaSki respectively, while a further 100% reduction of DVL3 was observed at 12 and 24 hours for both cell lines. (E) Upon treatment of metformin, 60–80%reduction of DVL3 was observed in Hela, SiHa, CaSki, C41 and C33A in a dose-dependent manner.</p

Ectopic expression of DVL3 enhances Wnt/β-catenin signaling activity and cell proliferation in cervical cancer cells.

<p>(A) Western blot showed that stably over-expressing GFP-DVL3 elevated expression of β-catenin in C33A (C-G25 and C-G28) and SiHa (S-G18 and S-G20). (B) XTT assay revealed a significant increase of cell proliferation in cervical cancer cells with stable expression of GFP-DVL3 (C-G24 and C-G28; <i>P</i><0.05) (S-G18 and S-G20; <i>P</i><0.05). (C) Clonogenic assay showed that twice and thrice the number of colonies were found in GFP-DVL3 ectopically expressing C33A cells (C-G25 and C-G28) (<i>P</i><0.05) and SiHa cells (S-G18 and S-G20) (<i>P</i><0.05) as compared with their vector controls.</p

Clinicopathological correlation of DVL3 expression in cervical cancer tissue array (CX1021) (Pantomics, CA, USA).

<p>Clinicopathological correlation of DVL3 expression in cervical cancer tissue array (CX1021) (Pantomics, CA, USA).</p

AMPK activators reduce DVL3 through either inactivation of mTOR activity or increased proteasomal degradation in cervical cancer cells.

<p>(A) Rapamycin inactivated p70S6 kinase activity and aborted protein synthesis of DVL3 in all cervical cancer cell lines (C33A, C41, CaSki, HeLa and SiHa) in a dose-dependent manner. (B) Proteasomal inhibitor, AM114, restored the reduced DVL3 mediated by metformin in C33A. (C) Proteasomal inhibitor, AM114, could not restore the reduced DVL3 mediated by metformin in SiHa and HeLa. (D) <i>In vivo</i> ubiquitination assay showed that DVL3 was degraded as poly-ubiquitinated DVL3 protein ((Ub)n-DVL3) in C33A cell model. C33A cells were transfected with pcDNA-HA(Ub)₈ plasmid. Both MG132 or AM114 and metformin were used to enhance the amount of polyubiquitinated DVL3 protein products ((Ub)n-DVL3). Cell lysates were incubated with anti-DVL3, and the immunoprecipitates were probed with anti-HA. The same membrane was re-probed with anti-DVL3 to detect the expression of DVL3.</p