Product Focus: CellTiter-Glo™ Luminescent Cell Viability Assay

Inhibition of Ligand-mediated HER2 Activation in Androgen-independent Prostate Cancer

This paper investigated the role of HER2 tyrosine kinase overexpression and activation in the development of androgen independence in prostate cancer. Previous studies have provided conflicting results about HER2 expression. In order to resolve these conflicts, the authors studied the AI prostate cancer cell line, AI 22Rv1. Using Western analysis, they determined that this cell line expresses HER2, EGFR and HER3. (HER2 forms active heterodimers with HER3 and EGFR.)

The authors investigated heregulin activation of HER2 in these cells using the CellTiter-Glo™ Luminescent Cell Viability Assay (Cat.#  G7570, G7571, G7572, G7573). Cells were seeded into black, 96-well plates and allowed to adhere overnight. The next day the cells were serum starved for 24 hours and then treated with different concentrations of rhuMAb C24 (an inhbitor of HER2 activation) for 1 hour followed by treatment with heregulin or TNFα. Cells were incubated 4 days before measuring cell proliferation.

Cells treated with heregulin showed significant cell proliferation compared to control cells while TNFα did not induce the same proliferation. The inhibitor antibody rhuMAb inhibited heregulin-induced proliferation in a dose-dependent manner.

Mendoza, N., Lewis, G.L., Silva, J., Schwall, R., and Wickramasinghe, D.^1* (2002) Can. Res. 62, 5485–8.

¹Molecular Oncology, Genetech Inc., South San Francisco, California 94080

*To whom correspondence should be addressed. 

Product Focus: CellTiter-Glo™ Luminescent Cell Viability Assay

Action of a Novel Anticancer Agent, CHS 828, on Mouse Fibroblasts: Increased Sensitivity of Cells Lacking Poly (ADP-Ribose) Polymerase-1

These authors investigated the role of PARP-1 in modulating cellular response to the anti-cancer drug, CHS 828. The primary mechanism of CHS 828 toxicity is unknown. However, some investigations have shown that it can induce an increase in extracellular acidification rate, suggesting that the drug may affect mitochondrial respiration. To investigate this possibility, immortalized embryonic fibroblasts were obtained from PARP-1-/- mice. The effect of CHS 828 on proliferation of these cells was assessed by two methods. One method detected the conversion of a tetrazolium salt to a colored formazan product, reflecting the reducing potential of the cell. Since CHS 828 may affect mitochondria, the authors also used the CellTiter-Glo™ Luminescent Cell Viability Assay (Cat.#  G7570, G7571, G7572, G7573) to assess cell viability. This assay correlates the amount of ATP in the cells with cell viability. Both assays showed that growth was inhibited in mouse fibroblasts lacking PARP-1 when treated with CHS 828 compared to untreated controls. Human PARP-1 expression conferred resistance to the drug in these cells.

Lövborg, H.¹, Wojciechowski, J.², Larson, R.¹, Węsierska-Gądeck, J.^2* (2002) Cancer Research 62, 4206–11.

¹Department of Clinical Pharmacology, Uppsala University Hospital, Uppsala, Sweden
²Institute of Cancer Research, University of Vienna, Vienna, Austria
*To whom correspondence should be addressed. 
Jozefa.Antonia.Gadek-Wesierski@univie.ac.at 

Product Focus: Dual-Luciferase^® Reporter Assay System

Anti-TAR Polyamide Nucleotide Analog Conjugated with a Membrane-Permeating Peptide inhibits Human Immunodefficiency Virus Type 1 Production

 Kaushik, N.^1,2 , Basu, A.^1,2, Palumbo, P.³, Myers, R.L.⁴, Pandey, V.N.^1,2* (2002) J. Virol. 76, 3881–91


High mutation rates in HIV-1, particularly in the reverse transcriptase and protease genes, have complicated control of HIV through chemotherapy. To circumvent this problem, researchers have focused on regions of the viral genome that are relatively well conserved and not as prone to mutational variation. One such region is the HIV 5' LTR which contains a number of elements that are essential for viral replication. The TAR (Transactivation Response) is one such essential element. It forms a RNA stem-loop structure and is transcriptionally activated by the protein Tat. The precise regions of the TAR stem-loop that are involved in interacting with Tat are well characterized. Point mutations that destabilize the stem of TAR, result in subsequent premature termination of viral transcription. Thus the Tat/TAR elements have become attractive targets for AIDS drug design. 

A number of therapeutic strategies specifically target Tat/TAR including: (1) hindering this interaction using Tat analogs; (2) directly disrupting the stem-loop structure itself; and (3) using oligonucleotide decoys that sequester the Tat and make it unavailable for TAR binding. This paper describes a novel approach involving PNA (polyamide nucleotide analog). Because of its peptide bond/nucleotide base composition, PNA is resistant to both protease and nuclease degradation. By constructing an anti-TAR PNA that is specific and complementary to a TAR sequence, these researchers were able to inhibit Tat-mediated transactivation. Moreover, they conjugated the PNA to a molecule called Transportan to aid in cell permeabilization. Through gel shift assays, these researchers showed that the transportan conjugated anti-TAR PNA efficiently bound to its TAR target sequence. Additionally, the anti TAR PNA effectively inhibited reverse transcription across TAR. 

The authors used the Dual-Luciferase^® Reporter Assay System (Cat.# E1910, E1960) and the pRLCMV Vector (Cat.# E2261) on cell cultures to demonstrate inhibition of HIV-LTR promoter-driven expression by the anti-TAR PNA-Transportan conjugate. RNase protection assays showed that such inhibition occurred at the transcriptional level of LTR-driven gene expression. Finally, studies of the p24 antigen (a marker of HIV viral titer) in cultured H9 cells that were chronically infected by HIV, revealed that the anti-TAR PNA-Transportan conjugate significantly reduced viral titers. ELISA-based tests, for example, showed up to a 70–90% decrease in p24 antigen levels with anti TAR PNA-Transportan treatment.

¹Center for the Study of Emerging and Re-Emerging Pathogens, UMDNJ-New Jersey Medical School Newark, N.J.
²Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School Newark, N.J.
³Division of Allergy, Immunology and Infectious Diseases, Dept. of Pediatrics, UMDNJ-New Jersey Medical School Newark, N.J.
⁴Applied Biosystems, Framingham, MA.

*To whom correspondence should be addressed. 
pandey@umdnj.edu

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