Autofluorescent proteins as photosensitizer in eukaryontes

Abstract

Since the discovery of the green fluorescent green protein (GFP) in 1961 many variants of fluorescent proteins (FP) were detected. The importance was underlined by the Nobel price award in chemistry 2008 for the invention, application, and development of the GFP by Shimomura, Chalfie and Tsien. GFP, first described by Shimomura now is indispensible in the scientific daily life. Since then and also in future fluorescent proteins will lead to new applications as reporters in cell biology. Such FPs can absorb visible day-light and predominantly one variant of the red fluorescent protein, the KillerRed protein (KRED) emits active electrons producing reactive oxygen species (ROS) leading to photokilling processes in eukaryotes. KRED can be activated by daylight as a photosensitizing agent. It is quite obvious that the KRED's expression and localization is critical with respect to damage, mutation and finally killing of eukaryotic cells. We found evidence that the KRED's cytotoxicity is ascendantly location-dependent from the cell membrane over the nuclear lamina to the chromatin in the cell nucleus. Daylight illumination of cells harbouring the KRED protein fused with the histone H2A, a DNA-binding protein which is critical for the formation of the chromatin structure results in cell killing. Therefore the H2A-KRED fusion protein can be considered as an appropriate candidate for the photodynamic therapy (PDT). This finding can be transferred to current photodynamic approaches and can enhance their therapeutic outcome.

Keywords: KillerRed; Melanoma; Photo-Dynamic-Therapy (PDT); ROS; Skin Tumors; fluorescent Proteins; subcellular Localization; topical Application.

Figure 1

The figure exemplifies the generation of ROS and their influence on downstream targets in vascular cells. ROS influence a multifaced range of cellular activities e.g. of protein tyrosine phosphatases (PTP). ROS influence gene and protein expression by activating transcription factors, such as NFκB and AP-1. ROS stimulate Ca²⁺ channels leading to increased [Ca²⁺]. ROS influence matrix metalloproteinases (MMPs), modulating extracellular matrix protein (ECM) degradation. (Modified according to Touyz and Filomeni) [Touyz, R. M. Antioxid. Redox. Signal. 2005, 7 (9-10), 1302-1314; Filomeni, G.; et al. Biochem. Pharmacol. 2002, 64 (5-6), 1057-1064].

Figure 2

The figure displays the physical map of the pEGFP a red-shifted variant of the WT GFP optimized for higher fluorescence and higher expression in mammalian cells. GenBank Acc. No.: #U55762. (Details see Clontech user manual http://www.clontech.com/images/pt/dis_vectors/PT3027-5.pdf)

Figure 3

This physical map shows the body of the pKillerRed mammalian expression vector encoding the red fluorescent protein KillerRed alone in eukaryotic (mammalian) cells (Evrogen FP961; GenBank Acc. No.: AY969116). (Details see Evrogen user manual http://www.evrogen.com/products/KillerRed/KillerRed_Related_products.shtml)

Figure 4

The figure illustrates the pKillerRed-mem a mammalian expression vector which encodes membrane-targeted KillerRed (Cat. No.: #FP966). (Details see Evrogen user manual http://www.evrogen.com/products/KillerRed/KillerRed_Related_products.shtml) shows the mem sequence.

Figure 5

Physical map of the vector expressing the fusion protein KRED-Lamin B1. The Lamin B1 was inserted into the MCS. (The Lamin B1 sequence was kindly provided by Harald Herrmann, this institute)

Figure 6

Physical map of the vector expressing the histone fusion protein H2A-KRED. The histone H2A was inserted into the MCS.

Figure 7

The graph demonstrates the influence of the illumination time on the cellular phenotype and displays the relative number of morphologically intact HeLa cells.

Figure 8

HeLa cells (left) and DU 145 (right) stably transfected with pKillerRed-mem vector encoding membrane-targeted KillerRed. The bars represent 25 µm (bottom right corner).

Figure 8

HeLa cells (left) and DU 145 (right) stably transfected with pKillerRed-mem vector encoding membrane-targeted KillerRed. The bars represent 25 µm (bottom right corner).

Figure 9

HeLa cells (left) and DU 145 cells (right) stably transfected with the pKillerRed Lamin B1 vector expressing the fusion protein KRED-Lamin B1. The bars represent 25 µm (bottom right corner)

Figure 9

HeLa cells (left) and DU 145 cells (right) stably transfected with the pKillerRed Lamin B1 vector expressing the fusion protein KRED-Lamin B1. The bars represent 25 µm (bottom right corner)

Figure 10

HeLa cells (left) and DU 145 cells (right) stably transfected with the pH2A-KillerRed vector expressing the fusion protein histone H2A-KRED located in the cell nuclei. The bars represent 10 µm (bottom right corner).

Figure 10

HeLa cells (left) and DU 145 cells (right) stably transfected with the pH2A-KillerRed vector expressing the fusion protein histone H2A-KRED located in the cell nuclei. The bars represent 10 µm (bottom right corner).

Figure 11

HeLa and DU 145 cells stably transfected with pKRED-mem and pKRED-Lamin to different cellular locations. Morphologically intact cells were counted after the illumination's time points: 30, 60, 90, 120, 180 minutes.