Marker Gene Monthly Newsletter - Volume 11, Number 6 - June, 2011

Cellular Senescence Induced by Adriamycin

Cellular senescence is a condition in which cells stop dividing and proliferation slows or stops. Many primary cell types exhibit this limited capacity to reproduce in cell culture, typically undergoing between 40 and 60 cell divisions, but then becoming senescent. When cells become senescent changes in morphology and gene expression patterns occur. But senescence can also be induced in cells in response to stress mechanisms that are manifested by telomere shortening and DNA damage. Although application of high levels of cytotoxic drugs cause cell death, typically through necrosis, lower levels of damage can determine whether programmed cell death (apoptosis) or senescence will occur. Sub-cytotoxic levels of drugs will typically induce senescence. Senescence induced by DNA-damaging anticancer drugs provide one of the key modes of action in chemotherapy, since cancer cells are often resistant to initiating apoptosis in response to drug action. These drug-induced senescent cells will exhibit an increase in SA-b-Gal (Senescence-Associated b-Galactosidase) activity as well as induction of polyploidization and DNA damage.

Recent work from the laboratory of Dr. Yuji Nakayama and co-workers at the Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University measured senescence associated morphology changes in HeLa cell lines induced by sub-toxic levels of the chemotherapeutic drug adriamycin. In addition to induction of polyploidization and senescence-associated morphology changes, such as flattened and enlarged cell shape, the induction of b-galactosidase activity was measured as a function of drug levels. The drug was also found to cause DNA damage-induced decreases in the levels of cyclin B, a regulatory protein involved in mitosis.

To measure SA-b-gal activity, adriamycin-treated cells (20ng/3days) were incubated with the substrate Fluorescein di-b-D-Galactopyranoside (FDG, M0250) (20 uM), an ultrasensitive substrate for b-galactosidase activity. FDG gives rise to fluorescence after being cleaved by b-galactosidase, enabling detection of SA-b-gal activity by fluorescent microscopy and flow cytometry. Interestingly, gated cytometric data also correlated SA-b-Gal activity with increased cell size, which is known to be a mophology change associated with senescence. Marker Gene now provides a convenient kit for measurement of SA-b-Gal activity in senescence (MarkerGene™ Cellular Senescence Microtiterplate Assay Kit, M1405) which also includes standards and control X-Gal analysis systems. For more information about these techniques, please see the references below or visit our website.

• Kikuchi I, Nakayama Y, Morinaga T, Fukumoto Y, Yamaguchi N (2010) "A decrease in cyclin B1 levels leads to polyploidization in DNA damage-induced senescence." Cell Biol. Int. 34: 645–653.
• Nolan GP, Fiering S, Nicolas JF, Herzenberg LA (1988) "Fluorescence-activated cell analysis and sorting of viable mammalian cells based on b-D-galactosidase activity after transduction of Escherichia coli lacZ." Proc. Natl. Acad. Sci. USA 85:.2603-2607.
• Collado M, Blasco MA, Serrano M (2007) "Cellular senescence in cancer and aging." Cell 130:223–33.
• Mashal RD, Lester S, Corless C, Richie JP, Chandra R, Propert KJ, Dutta A, (1996) "Expression of cell cycle-regulated proteins in prostate cancer." Cancer Res. 56(18): 4159-4163.
• d’Adda di Fagagna F. (2008) "Living on a break: cellular senescence as a DNA-damage response." Nature Rev. Cancer 8: 512–522.
  
• Roninson IB. (2003) "Tumor cell senescence in cancer treatment." Cancer Res.63:2705–15.
  
• Brown JM, Wouters BG. (1999) "Apoptosis, p53, and tumor cell sensitivity to anticancer agents." Cancer Res 1999;59:1391-1399.
  

Bacterial Multiple Drug Resistance Detection

Multiple Drug Resistance in bacterial cell strains continues to represent a major problem for clinical control of disease and infection. In particular, multiple drug resistance in Pseudomonas aeruginosa (MDRP) to major drug classes, including the carbapenems, quinolones, and aminoglycosides has been the cause of several major infection outbreaks. P. aeruginosa has evolved to have a number of intrinsic resistance mechanisms to common antibiotics, including several cell surface efflux pumps (RND pumps) that can effectively translocate these drugs out of the cells. In particular the MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY efflux systems are known to have important roles in multiple drug resistance . Because of the paucity of new antibacterial agents for P. aeruginosa, RND pump inhibitors have become increasingly more attractive as potential auxiliary agents for treating MDRP infections. One such efflux pump inhibitor, Phe-Arg-b-naphthylamide (PAbN, MC-207,110) has shown promise in combination with several antibiotics, although no clinically useful inhibitor is currently available.

Recent work from the laboratory of Professors Ryota Iino, Yoshimi Matsumoto and co-workers at Osaka University and the University of Tokyo (Japan) have developed a new method to evaluate efflux-inhibitory activities of several efflux proteins in E. coli using the fluorogenic substrate FDG and a microfluidic channel device. Fluorescein-di-b-D-galactopyranoside (FDG, M0250) is a fluorogenic compound that is non-fluorescent until it is hydrolyzed by b-galactosidase in the cytoplasm of Escherichia coli to produce a bright fluorescent dye fluorescein. The group found that both FDG and fluorescein were substrates of the efflux pumps, and were able to evaluate the efficacy of two inhibitors; the MexB-specific pyridopyrimidine (D13-9001) and non-specific inhibitor Phe-Arg-b-naphthylamide (PAbN) in combination with several drug candidates toward a variety of efflux pump systems in the bacteria. Application of these methods to the discovery of additional efflux pump inhibitors in bacterial cell strains like Pseudomonas aeruginosa are significant. To find out more about these methods , please see references below or visit our website.

• Lister PD, Wolter DJ, Hanson ND (2009) "Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded
  resistance mechanisms." Clin Microbiol Rev 22: 582–610.
• Matsumoto Y, Hayama K, Sakakihara S, Nishino K, Noji H, et al. (2011) "Evaluation of Multidrug Efflux Pump Inhibitors by a New Method Using Microfluidic Channels." PLoS ONE 6(4): e18547. doi:10.1371/journal.pone.0018547.
• Yoshida K, Nakayama K, Ohtsuka M, Kuru N, Yokomizo Y, Sakamoto A, Takemura M, Hoshino K, Kanda H, Nitanai H, Namba K, Yoshida K, Imamura Y, Zhang JZ, Lee VJ, Watkins WJ. (2007) " MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 7: highly soluble and in vivo active quaternary ammonium analogue D13-9001, a potential preclinical candidate." Bioorg. Med Chem. 15: 7087–7097.
  

Mammalian Cell Multiple Drug Resistance Assays

Multiple drug resistance in mammalian cells is characterized by over-expression of membrane transport proteins ABCG2 and ABCB1 that induce efflux of anticancer drugs out of the cell and subsequently cause tumor cell resistance to those drugs. Overcoming MDR has become a major challenge for chemotherapy. Early detection of drug resistant tumor cells may be critical in successful treatment of tumors. In addition, multiple drug resistance has become important to monitor in treatment of several other diseases such as tuberculosis, cholera, malaria and HIV-AIDS. Traditionally MDR has only been detectable using time-consuming assays involving the growth of cells in media containing the drug and assessing the level of cell death.

The MarkerGene™ Multiple Drug Resistance Microtiterplate Assay Kit (M1580) is a high-throughput assay system based upon measurement of efflux of the fluorescent dye Rhodamine 123 (R123).  Rhodamine 123 is known to bind to the MDR transporters ABCG2 and ABCB1 in a manner similar to many chemotherapeutic drugs. The efflux of this dye can be measured by its fluorescence in the media (EM 504nm: EX 538nm).  The change of efflux is evaluated by comparing R123 transport into the medium over a series of time points. Cells exhibiting multiple drug resistance display a rapid increase in efflux of R123 over time, while non-resistant cells display a steady efflux. For more information about this new kit, please see the references below, or visit our website.

• Harker WG, MacKintosh FR, Sikic BI (1983) "Development and Characterization of a Human Sarcoma Cell Line, MES-SA, Sensitive to Multiple Drugs" Cancer Research 43(10): 4943-4950.
• Harker WG, Sikic BI, (1985) "Multidrug (Pleiotropic) Resistance in Doxorubicin-selected Variants of the Human Sarcoma Cell Line MES-SA" Cancer Research 45(9): 4091-4096.
• Kessel D, Beck WT, Kukuruga D, Schulz V, (1991) "Characterization of Multidrug Resistance by Fluorescent Dyes" Cancer Research 51(17): 4665-4670.
• Ludescher C, J Drach J, Hofmann J, Grunicke H, (1991) "Rapid functional assay for the detection of multidrug-resistant cells using the fluorescent dye rhodamine 123." Blood 78(5): 1385-1387.
• Altenberg GA, Vanoye CG, Horton JK, Reuss L (1994) "Unidirectional fluxes of rhodamine 123 in multidrug-resistant cells: evidence against direct drug extrusion from the plasma membrane." PNAS USA 91(11): 4654-4657.
• Wesolowska O, Paprocka M, Kozlak J, Motohashi N, Dus D, Michalak K.(2005) "Human sarcoma cell lines MES-SA and MES-SA/Dx5 as a model for multidrug resistance modulators screening." Anticancer Research 25 1A:383-389.

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