Isolation of two cDNAs encoding novel α1-antichymotrypsin-like proteins in a murine chondrocytic cell line

Inglis, J.M.; Lee, Muriel; Davidson, Duncan; Hill, Robert E.

doi:10.1016/0378-1119(91)90201-l

Cited by 43 publications

(28 citation statements)

References 31 publications

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“…The transforming activity of the antisense Spi-2 cDNA was presumably due to an inhibitory effect on the stability or translation of mRNAs encoding the Spi-2 protease inhibitor (20), leading to increased secreted protease activity and disruption of cell-cell or cell-matrix contacts which normally repress proliferation.…”

Section: Results and Discussion Design Of Retroviral Vectorsmentioning

confidence: 99%

Expression Cloning of Oncogenes by Retroviral Transfer of cDNA Libraries

Whitehead

Kirk

Kay

1995

Molecular and Cellular Biology

171

148

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A cDNA library transfer system based on retroviral vectors has been developed for expression cloning in mammalian cells. The use of retroviral vectors results in stable cDNA transfer efficiencies which are at least 100-fold higher than those achieved by transfection and therefore enables the transfer and functional screening of very large libraries. In our initial application of retroviral transfer of cDNA libraries, we have selected for cDNAs which induce oncogenic transformation of NIH 3T3 fibroblasts, as measured by loss of contact inhibition of proliferation. Nineteen different transforming cDNAs were isolated from a total of 300,000 transferred cDNA clones. Three of these cDNAs were derived from known oncogenes (raf-1, lck, and ect2), while nine others were derived from genes which had been cloned previously but were not known to have the ability to transform fibroblasts (␤-catenin, thrombin receptor, phospholipase C-␥ 2 and Spi-2 protease inhibitor genes). The Spi-2 cDNA was expressed in antisense orientation and therefore is likely to act as an inhibitor of an endogenous transformation suppressor. Seven novel cDNAs with transforming activities, including those for three new members of the CDC24 family of guanine nucleotide exchange factors, were also cloned from the retroviral cDNA libraries. Retroviral transfer of libraries should be generally useful for cloning cDNAs which confer selectable phenotypes on many different types of mammalian cells.Cellular oncogenes or proto-oncogenes can be cloned by selecting for their ability to confer the phenotype of deregulated growth on cells in which they are expressed. This was first done by transfecting fragmented genomic DNA into cell lines such as NIH 3T3 fibroblasts which are susceptible to single-hit oncogenic transformation (13,15,31,36,40,46). Subsequently, stable transfection of these cells with cDNA libraries in plasmid or phage expression vectors has been used as an alternative approach, to avoid the severe difficulties which have been encountered in recovering and analyzing oncogenes present in transfected genomic DNA (8,9,[32][33][34]. Despite the theoretical advantages of working with transfected cDNA expression libraries, only a small fraction of the many currently known oncogenes have been cloned in this way. The most significant limitation in the use of cDNA library transfer for cloning oncogenes has been the low efficiencies of cDNA transfer and expression which can be achieved by stable transfection methods. It is difficult to generate more than a few tens of thousands of transfectants in which cDNA clones are being expressed at adequate levels, but a comprehensive screening of a mammalian cDNA library demands the transfer and expression of several million clones. Therefore, the small number of oncogenes which have been cloned so far from transferred libraries is unlikely to be due to exhaustion of the pool of cDNAs with oncogenic potential but rather is a result of a failure to transfer, and thus detect, most such cDNAs.In contrast to deliberatel...

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Section: Results and Discussion Design Of Retroviral Vectorsmentioning

confidence: 99%

Expression Cloning of Oncogenes by Retroviral Transfer of cDNA Libraries

Whitehead

Kirk

Kay

1995

Molecular and Cellular Biology

171

148

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“…The cDNA for spi2a was originally cloned from a chondrocyte cell line (6) and subsequently demonstrated to be expressed in hemopoietic progenitor cells (7). Interestingly, Hampson et al (7) found spi2a mRNA to be down-regulated during granulocyte macrophage differentiation of a multipotential hemopoietic progenitor cell line and during macrophage differentiation of bipotential granulocyte/macrophage precursor cells isolated from mouse bone marrow.…”

Section: Discussionmentioning

confidence: 99%

“…Using cDNA arrays, we investigated the changes in gene expression associated with macrophage activation during in vivo infection of mice with the intracellular bacterium Mycobacterium bovis bacillus Calmette-Guérin (BCG), 4 a classical system for studying immune macrophage activation. In this screen, we identified serpin 2a (spi2a) as a protein with substantially increased expression during BCG infection in vivo (6,7). Serpins are a protein superfamily with conserved structure that regulate both serine and cysteine protease function in diverse processes including coagulation, extracellular matrix degradation, complement activation, fibrinolysis, and apoptosis (8,9).…”

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confidence: 99%

Serpin 2a Is Induced in Activated Macrophages and Conjugates to a Ubiquitin Homolog

Hamerman

Hayashi

Schroeder

et al. 2002

The Journal of Immunology

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After i.p. infection of mice with the intracellular bacterium Mycobacterium bovis bacillus Calmette-Guérin, macrophages recovered from the peritoneal cavity display classical signs of immune activation. We have identified a member of the serine protease inhibitor (serpin) family which is highly induced in macrophages during bacillus Calmette-Guérin infection. Serpin 2a (spi2a) expression is also induced in macrophages in vivo during infection with Salmonella typhimurium and Listeria monocytogenes, and in vitro by a variety of bacteria and bacterial products. The cytokine IFN-γ also induces spi2a expression in macrophages, and this induction is synergistic with bacterial products. We also demonstrate here that a ubiquitin homolog, IFN-stimulated gene of 15-kDa (ISG15), is strongly induced during in vitro and in vivo activation of macrophages and that it conjugates to spi2a in activated macrophages. The ISG15-spi2a conjugates were identified by tandem mass spectrometry and contained spi2a conjugated to either one or two molecules of ISG15. Whereas spi2a was induced by either bacterial products or IFN-γ, ISG15 was induced only by bacterial products. Although many protein targets have been described for ubiquitin conjugation, spi2a is the first ISG15-modified protein to be reported. Macrophage activation is accompanied by the activation of a variety of proteases. It is of interest that a member of the serine protease inhibitor family is concomitantly induced and modified by a ubiquitin-like protein.

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“…Complete inhibition of caspase activity was verified by enzyme assay [14]. The level of Spi2A mRNA was quantitated by real-time PCR using primers and probes specific for Spi2A [12] and cyclofilin A control mRNA [16], either before (Fig. 1B) or 4 h after ( Fig.…”

Section: Tnf-a Death Assaysmentioning

confidence: 99%

Serine protease inhibitor 2A inhibits caspase‐independent cell death

2004

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The release of cysteine cathepsins from the lysosome into the cytoplasm can trigger programs of cell death (PCD) that do not require caspase executioner proteases but instead are mediated by toxic reactive oxygen species (ROS). Here, we show that a cytoplasmic inhibitor of papain-like cathepsins -Serine protease inhibitor 2A (Spi2A) -is required for the protection of cells from caspase-independent PCD triggered by tumor necrosis factor-. In the absence of caspase activity, Spi2A suppressed PCD by inhibiting cathepsin B after it was released into the cytoplasm. Spi2A also directly protected against ROS-mediated PCD, which is consistent with a role in suppressing caspaseindependent pathways of PCD. We conclude that inhibition of lysosomal executioner proteases by Spi2A is a physiological mechanism by which cells are protected from caspase-independent programmed cell death.

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Isolation of two cDNAs encoding novel α1-antichymotrypsin-like proteins in a murine chondrocytic cell line

Cited by 43 publications

References 31 publications

Expression Cloning of Oncogenes by Retroviral Transfer of cDNA Libraries

Expression Cloning of Oncogenes by Retroviral Transfer of cDNA Libraries

Serpin 2a Is Induced in Activated Macrophages and Conjugates to a Ubiquitin Homolog

Serine protease inhibitor 2A inhibits caspase‐independent cell death

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