A tumour suppressor network relying on the polyamine-hypusine axis

Tumour suppressor genes encode a broad class of molecules whose mutational attenuation contributes to malignant progression. In the canonical situation, the tumour suppressor is completely inactivated through a two-hit process involving a point mutation in one allele and chromosomal deletion of the other. Here, to identify tumour suppressor genes in lymphoma, we screen a short hairpin RNA library targeting genes deleted in human lymphomas. We functionally identify those genes whose suppression promotes tumorigenesis in a mouse lymphoma model. Of the nine tumour suppressors we identified, eight correspond to genes occurring in three physically linked 'clusters', suggesting that the common occurrence of large chromosomal deletions in human tumours reflects selective pressure to attenuate multiple genes. Among the new tumour suppressors are adenosylmethionine decarboxylase 1 (AMD1) and eukaryotic translation initiation factor 5A (eIF5A), two genes associated with hypusine, a unique amino acid produced as a product of polyamine metabolism through a highly conserved pathway. Through a secondary screen surveying the impact of all polyamine enzymes on tumorigenesis, we establish the polyamine-hypusine axis as a new tumour suppressor network regulating apoptosis. Unexpectedly, heterozygous deletions encompassing AMD1 and eIF5A often occur together in human lymphomas and co-suppression of both genes promotes lymphomagenesis in mice. Thus, some tumour suppressor functions can be disabled through a two-step process targeting different genes acting in the same pathway.     

Fbxw7/Cdc4 is a p53-dependent, haploinsufficient tumour suppressor gene

The FBXW7/hCDC4 gene encodes a ubiquitin ligase implicated in the control of chromosome stability. Here we identify the mouse Fbxw7 gene as a p53-dependent tumour suppressor gene by using a mammalian genetic screen for p53-dependent genes involved in tumorigenesis. Radiation-induced lymphomas from p53+/- mice, but not those from p53-/- mice, show frequent loss of heterozygosity and a 10% mutation rate of the Fbxw7 gene. Fbxw7+/- mice have greater susceptibility to radiation-induced tumorigenesis, but most tumours retain and express the wild-type allele, indicating that Fbxw7 is a haploinsufficient tumour suppressor gene. Loss of Fbxw7 alters the spectrum of tumours that develop in p53 deficient mice to include a range of tumours in epithelial tissues such as the lung, liver and ovary. Mouse embryo fibroblasts from Fbxw7-deficient mice, or wild-type mouse cells expressing Fbxw7 small interfering RNA, have higher levels of Aurora-A kinase, c-Jun and Notch4, but not of cyclin E. We propose that p53-dependent loss of Fbxw7 leads to genetic instability by mechanisms that might involve the activation of Aurora-A, providing a rationale for the early occurrence of these mutations in human cancers.     

Identification of human brain tumour initiating cells

The cancer stem cell (CSC) hypothesis suggests that neoplastic clones are maintained exclusively by a rare fraction of cells with stem cell properties. Although the existence of CSCs in human leukaemia is established, little evidence exists for CSCs in solid tumours, except for breast cancer. Recently, we prospectively isolated a CD133+ cell subpopulation from human brain tumours that exhibited stem cell properties in vitro. However, the true measures of CSCs are their capacity for self renewal and exact recapitulation of the original tumour. Here we report the development of a xenograft assay that identified human brain tumour initiating cells that initiate tumours in vivo. Only the CD133+ brain tumour fraction contains cells that are capable of tumour initiation in NOD-SCID (non-obese diabetic, severe combined immunodeficient) mouse brains. Injection of as few as 100 CD133+ cells produced a tumour that could be serially transplanted and was a phenocopy of the patient's original tumour, whereas injection of 10(5) CD133- cells engrafted but did not cause a tumour. Thus, the identification of brain tumour initiating cells provides insights into human brain tumour pathogenesis, giving strong support for the CSC hypothesis as the basis for many solid tumours, and establishes a previously unidentified cellular target for more effective cancer therapies.     

p15Ink4b is a critical tumour suppressor in the absence of p16Ink4a

The CDKN2b-CDKN2a locus on chromosome 9p21 in human (chromosome 4 in mouse) is frequently lost in cancer. The locus encodes three cell cycle inhibitory proteins: p15INK4b encoded by CDKN2b, p16INK4a encoded by CDKN2a and p14ARF (p19Arf in mice) encoded by an alternative reading frame of CDKN2a (ref. 1). Whereas the tumour suppressor functions for p16INK4a and p14ARF have been firmly established, the role of p15INK4b remains ambiguous. However, many 9p21 deletions also remove CDKN2b, so we hypothesized a synergistic effect of the combined deficiency for p15INK4b, p14ARF and p16INK4a. Here we report that mice deficient for all three open reading frames (Cdkn2ab-/-) are more tumour-prone and develop a wider spectrum of tumours than Cdkn2a mutant mice, with a preponderance of skin tumours and soft tissue sarcomas (for example, mesothelioma) frequently composed of mixed cell types and often showing biphasic differentiation. Cdkn2ab-/- mouse embryonic fibroblasts (MEFs) are substantially more sensitive to oncogenic transformation than Cdkn2a mutant MEFs. Under conditions of stress, p15Ink4b protein levels are significantly elevated in MEFs deficient for p16Ink4a. Our data indicate that p15Ink4b can fulfil a critical backup function for p16Ink4a and provide an explanation for the frequent loss of the complete CDKN2b-CDKN2a locus in human tumours.     

Identification of specific binding proteins for a nuclear location sequence

The nuclear envelope is a selective barrier against the movement of macromolecules between the nucleus and cytoplasm. Nuclear proteins larger than relative molecular mass 20,000-40,000 are probably actively transported across the envelope through the nuclear pore complex and are directed by specific nuclear location sequences (NLS) in the proteins. NLS mediate the nuclear import of isolated nuclear proteins after microinjection into whole cells and the nuclear accumulation of chimaeric proteins or of non-nuclear proteins conjugated to synthetic peptides. The best-characterized NLS is the simian virus 40 large T-antigen sequence. We have identified two proteins of rat liver by chemical cross-linking that interact with a synthetic peptide containing this sequence: this interaction is specific for a functional NLS, is saturable, and high affinity. The binding proteins are present in a post-mitochondrial supernatant, in nuclei and in a nuclear envelope fraction, which is consistent with a role in the transport of nuclear proteins from the cytoplasm to the nucleus.     

Investigating the function of Akt by tet-off inducible expression system

A tet-off inducible cell line named BBT derived from BA/F3b cell line was constructed and the effect of this inducible expression system was significant when detected by tet-off responded luciferase reporter gene assay. Then tet-off responded Akt expression plasmid was transfected into BBT cells, and the stable cell lines were screened. The result of Northern blot showed that the expression of akt was significantly inducible. The clone with the best inducible effect was selected and named BBA for investigating the function of Akt. We found that Akt could significantly inhibit zinc-induced decrease of cell viability when assayed by MTT method. And the flow cytometric analysis showed that Akt could markedly repress zinc-induced apoptosis.     

组合径向基函数神经网络的硬件实现

为了提高径向基函数(RBF)神经网络的泛化能力,提出了一种组合径向基函数神经网络,并通过对英文字母的识别进行了仿真验证。基于CMOS电路设计了该组合径向基函数神经网络,所有单元电路均采用HJTC 0.18μm CMOS数模混合工艺设计制造。通过PCB板实现了一个2×3的组合RBF神经网络,并对"一"和"1"的识别问题进行了验证。实验结果表明:该组合RBF神经网络电路结构简单,便于扩展和调节,提高了整个网络的泛化能力,为硬件实现更为复杂的组合径向基函数神经网络提供了可能。