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Oral squamous cell carcinoma (OSCC) contributes to greater than 90% of all malignant tumors of the oral cavity. Treatment of OSCC patients often involves removing the lesion, much surrounding tissues and the lymph nodes of the neck. Despite the prevalence and the severity of OSCC, no relative tool to identify the progressive metastatic state of oral cancer cells exists. The authors of this study investigate whether loss of KAI1 expression, a putative metastasis suppressor gene originally identified in prostate cancers, could contribute to OSCC. In a study of precancerous lesions and OSCCs, the authors found no mutations in the KAI1 coding sequence, no loss of heterozygosity, and no hypermethylation of the KAI1 promoter region. However, immunohistochemical studies showed down-regulation of KAI1 protein expression in primary tumors. RT-PCR data derived from randomly selected primary OSCC specimens was consistent with the protein expression results.
The authors used Promega’s SV Total RNA Isolation System (Cat.# Z3100) to isolate total RNA form the randomly selected primary OSCC specimens and from paired specimens of noncancerous oral tissue. The total RNA was used in RT-PCR analysis of KAI1 expression.
Uzawa, K.1, Ono, K.1, Suzuki, H.2, Tanaka, C.3, Yakushiji, T.3, Yamamoto, N.3, Yokoe, H.1, Tanzawa, H.1* (2002) Clin. Cancer Res. 8, 828–835.
1Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan; 2Department of Urology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan; 3First Department of Oral and Maxillo-Facial Surgery, Tokyo Dental College, Chiba 261-8502, Japan.
*To whom correspondence should be addressed. tanzawap@med.m.chiba-u.ac.jp.
NF-κB is a transcription factor associated with genes controlling cell survival, proliferation, immune, and inflammatory responses. Normally inactive in the cell because of associations with inhibitory proteins (IκB), NF-κB is activated by the IκB kinase (IKK) signalsome. The IKK signalsome contains three major components: IKK1, IKK2, and NF-κB essential modulator (NEMO). NEMO is required for activation of NF-κB in response to proinflammatory cytokines. The human genetic disease, Incontinentia Pigmenti (IP), is caused by disruptions of the NEMO gene. Schmidt, et al. model human IP using a transgenic mouse NEMO knockout. Mice with the disrupted NEMO gene exhibit symptoms similar to human IP, which is characterized by X-linked lethality and unusual skin pigmentation patterns in heterozygous females. Homozygous NEMO-deficient embryos (NEMO–/–) at 12.5 days revealed liver degeneration resulting from apoptosis. Heterozygous mice (NEMO+/–) had increased heptatocyte apoptosis when compared to wild-type embryos. In addition to the increase liver apoptosis, the heterozygous mice developed skin pigmentation abnormalities reminiscent of the described skin pigmentation abnormalities of human IP.
Because inhibition of NF-κB activity sensitizes cells to TNF-induced apoptosis, the authors compared TNF cytotoxicity of NEMO–/–, IKK–/– and wildtype mouse embryonic fibroblasts (MEFs) using Promega’s CytoTox 96® Non-Radioactive Cytotoxicity Assay (Cat.# G1780). NEMO–/– MEFs are extremely sensitive to TNF cytotoxicity compared to IKK2–/– MEFs and wild-type MEFs under the same conditions. Additionally immunostaining using Promega’s Anti-PARP p85 Fragment pAb (Cat.# G7341), demonstrated increased apoptosis in the suprabasal skin layers in the affected areas of NEMO+/– mice. These results indicate that a combination of abnormal proliferation and increased apoptosis of keratinocytes lead to the skin phenotypes associated with the NEMO deficiencies.
Schmidt-Supprian, M.1*, Bloch W.2, Courtois, G.3, Addicks, K.2, Israel, A.3, Rajewsky, K.1 and Pasparakis, M.1 (2000) Mol. Cell 5, 981–992.
1Institute for Genetics, University of Cologne, D-50931 Cologne, Federal Republic of Germany; 2Department of Anatomy, University of Cologne, D-50931 Cologne, Federal Republic of Germany; 3Unité de Biologie Moléculaire, de lExpression Génique, URA 1773 CNRS, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
*To whom correspondence should be addressed. mrc@gmx.de
For many gene therapy applications the titer of retroviral vectors used to transport the gene(s) of interest into cells is a limiting factor, both in vitro and in vivo. Purification and concentration of virus from packaging cell supernatant can overcome this problem. In this study, Promega’s Streptavidin MagneSphere® Paramagnetic Particles (Cat.# Z5482) were used to concentrate and purify retrovirus particles from packaging cell supernatants.
Previous studies showed that retroviral particles released from murine fibroblast-derived PG13 packaging cells spontaneously complexed with formaldehyde-fixed Staphylococcus aureus (pansorbin). The binding to S. aureus coupled with the ability to maintain infectivity has been attributed to fibronectin binding proteins fnbA and fnbB from the Cowan I strain of S. aureus used in pansorbin. The fnbA and fnbB surface proteins may interact with fibronectin secreted by the NIH/3T3-derived packaging cells, the fibronectin, in turn, associating with the PG13-derived retrovirus. Thus retrovirus may interact with the fibronectin rather than directly with pansorbin. Fibronectin interactions are known to promote retroviral infectivity, a plausible explanation for the finding that retrovirus/pansorbin complexes remain infectious.
Despite the fact that pansorbin complexes well with retroviral vectors produced by NIH/3T3-derived packaging cells, it is less effective with other, non-NIH/3T3-derived packaging cell lines. As a poorly defined capture system is unlikely to be clinically useful, modifications were required to extend the range of applicable retroviral packaging cells as well as to improve reagent standardization. This paper studied three techniques involving the use of streptavidin-paramagnetic particles (SA-PMPs) for the concentration of retroviral particles.
In the first set of experiments, concentration of retroviral particles was accomplished using antibodies against murine fibronectin. SA-PMPs were conjugated with polyclonal rabbit anti-murine fibronectin antibodies and used to magnetically concentrate captured PG13-derived retrovirus, previously shown to complex with murine fibronectin. The authors used 1.25 × 109 PMPs to concentrate 5ml of retroviral particles; experiments using fewer PMPs resulted in crosslinking and clumping of the PMPs. The researchers determined that concentration with anti-mouse fibronectin antibodies conjugated to SA-PMPs (via protein A-biotin) greatly increased the amount of infectious retrovirus captured from cell supernatants. Retroviral titer increases of 2,000- to 3,000-fold occurred, bringing the total infectivity of captured retrovirus to 20X that of the control (nonconcentrated) sample; 1.4 × 105 cfu/ml for the control versus 4 × 108 cfu/ml for the anti-fibronectin concentrated samples.
A second approach sought to find a means of concentrating infective retroviral particles from packaging cells whose retrovirus progeny do not bind fibronectin. Affinity capture based on lectin was studied as an alternative for harvesting retroviral vectors from supernatants of such cells. The researchers also sought to extend this strategy to human HT1080-derived FLY packaging cells. By using lectin-binding PMPs, control titers of 4 × 105 (± 5.5 × 104) cfu/ml were increased to 7 × 108 (± 4 × 107) cfu/ml with biotin-BSI-B4 (Bandeiraea simplicifolia isolectin) or to 5.4 × 108 (± 5 × 107) cfu/ml with biotin-conA, after a 125-fold reduction in supernatant volume. Concentration by SA-PMPs alone resulted in a 4-fold increase in retroviral titers, demonstrating the improved efficiency of binding with lectin-PMPs. The lectin-PMP strategy was also tested on human HT1080-derived FLYA13- and FLYRD18-derived retrovirus, for which a successful concentration strategy had not previously been developed. While less effective for FLY- than for PG13-derived retrovirus, the PMP-lectin-mediated concentration is the most efficient described (at the writing of this article) for concentration of retrovirus from the family of FLY cells.
As the antibody- and lectin-mediated concentration strategies are neither universally applicable nor equally efficient, a third PMP retrovirus concentration strategy was designed. Based on a methodology used to label tumor and packaging cells, the PG13 packaging cell membranes were labeled with a succinimide ester to covalently couple biotin to cell-surface proteins. Biotinylation of the packaging cells results in the production of infectious biotinylated retrovirus particles, which are efficiently captured by SA-PMPs. In the absence of concentration, biotin- or carrier-treated particles do not increase retrovirus titers above control levels. Additionally, in the absence of biotinylation, the PMPs cannot capture and concentrate retrovirus. However, biotinylated packaging cells secreted retrovirus that coupled to streptavidin PMPs, allowing concentration to levels 4,200-fold above controls after a volume reduction of 125-fold.
The authors conclude that succinimide ester modification and biotin-mediated concentration of retroviral vectors may be universally applicable and highly efficient for use with all retroviral packaging cells. In addition the authors note that it is now possible to produce infectious, paramagnetic retroviral vector particles, which can be then directed to the desired location of infection by applying a magnetic field.
Hughes, C., Galea-Lauri, J., Farzaneh, F.* and Darling, D. (2001) Mol. Ther. 3, 623–30.
Guy’s, King’s, and St. Thomas’s School of Medicine, Department of Molecular Medicine, The Rayne Institute, 123 Coldharbour Lane, London, SE5 9NU, United Kingdom.
*To whom correspondence should be addressed: farzin.farzaneh@kcl.ac.uk
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