The therapeutic properties of light have been known for thousands of years, but it was only in the last century that photodynamic therapy (PDT) was developed. At present, PDT is being tested in the clinic for use in oncology--to treat cancers of the head and neck, brain, lung, pancreas, intraperitoneal cavity, breast, prostate and skin. How does PDT work, and how can it be used to treat cancer and other diseases?

Photodynamic therapy for cancer

Dynasore, a Cell-Permeable Inhibitor of Dynamin

Endocytosis in eukaryotic cells is characterized by the continuous and regulated formation of prolific numbers of membrane vesicles at the plasma membrane. These vesicles come in several different varieties, ranging from the actin-dependent formation of phagosomes involved in particle uptake, to smaller clathrin-coated vesicles responsible for the internalization of extracellular fluid and receptor-bound ligands. In general, each of these vesicle types results in the delivery of their contents to lysosomes for degradation. The membrane components of endocytic vesicles, on the other hand, are subject to a series of highly complex and iterative molecular sorting events resulting in their targeting to specific destinations. In recent years, much has been learned about the function of the endocytic pathway and the mechanisms responsible for the molecular sorting of proteins and lipids. This review attempts to integrate these new concepts with long-established views of endocytosis to present a more coherent picture of how the endocytic pathway is organized and how the intracellular transport of internalized membrane components is controlled. Of particular importance are emerging concepts concerning the protein-based signals responsible for molecular sorting and the cytosolic complexes responsible for the decoding of these signals.

/pdf/endocytosis-and-molecular-sorting-4gcwopp5ac.pdf

Endocytosis and molecular sorting

https://www.cell.com/molecular-cell/pdf/S1097-2765(01)00260-X.pdf

The Two-Handed E Box Binding Zinc Finger Protein SIP1 Downregulates E-Cadherin and Induces Invasion

A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells.

A transcriptional transactivator was developed that fuses the VP16 activation domain with a mutant Tet repressor from Escherichia coli. This transactivator requires certain tetracycline (Tc) derivatives for specific DNA binding. Thus, addition of doxycycline to HeLa cells that constitutively synthesized the transactivator and that contained an appropriate, stably integrated reporter unit rapidly induced gene expression more than a thousandfold. The specificity of the Tet repressor-operator-effector interaction and the pharmacological characteristics of Tc's make this regulatory system well suited for the control of gene activities in vivo, such as in transgenic animals and possibly in gene therapy.

https://science.sciencemag.org/content/sci/268/5218/1766.full.pdf

Transcriptional activation by tetracyclines in mammalian cells

Recently, we have described a regulatory system that allows the stringent control of individual gene activities in higher eukaryotic cell lines (1), in plants (2) and in animals (3,4). The essential components of this system are (i) an RNA polymerase II minimal promoter placed downstream of multiple operator sequences (tetO) of the Escherichia coli TnlO tetracycline resistance operon and (ii) a fusion between the Tet repressor (TetR) and the herpes simplex virus protein 16 (VP16), named tTA (1). In the absence of tetracycline (Tc), tTA binds to the tet operators to activate transcription from the minimal promoter, whereas in the presence of Tc its association and consequently its transcription activation is prevented. After the binding of tTA, minimal promoters derived from the cytomegalovirus IE promoter (PhCMv; 5) and fused to seven tetO sequences reach the remarkable strength of the parent promoter in HeLa cells when compared in transient expression assays (6). This high activation potential of tTA and the arrangement of its binding sites within PhCMV*-i [(!)'»see Fig. 1A] suggested the design of a bidirectional promoter which would allow the simultaneous regulation of two transcriptional units from centrally located multiple tetO sequences (Fig. 1A). Such a promoter should be useful for a number of experimental approaches. First, it may allow the co-regulation of the synthesis of two gene products in stoichiometric amounts, frequently a prerequisite for the production of heterodimeric (or hetero-oligomeric) proteins. Second, by fusing minimal promoters of differing efficiencies to the centrally located tetO sequences, two gene products may be co-regulated at different but defined levels. Third, by integrating an appropriate reporter gene at one side of the bidirectional promoter, the regulation of a not-readily-assayable gene of interest may be monitored via the reporter function. This latter possibility may also facilitate—at the cellular as well as at the organismal level—the screening for properly integrated expression units controlling the gene of interest.

/pdf/co-regulation-of-two-gene-activities-by-tetracycline-via-a-1q31yi1ytg.pdf

Co-regulation of two gene activities by tetracycline via a bidirectional promoter

Background

Tight control of gene activity has been achieved in cells and transgenic organisms using the Tet regulatory systems. Unregulated basal transcription can, however, be observed whenever integration of target genes driven by promoters responsive to tetracycline controlled transcriptional activators (tTA, rtTA) does not occur at suitable chromosomal sites. Moreover, in viral vectors containing both the tTA coding sequence and the regulated target gene, proximity of the enhancer element driving tTA/rtTA expression to the responsive unit will lead to elevated background levels. Similarly when tTA/rtTA responsive transcription units are in a non-integrated state as eg., during transient expression, intrinsic residual transcription persists in their ‘off’ state, which can differ in intensity among different cell types.



Methods

To efficiently repress such background activities we generated tetracycline controlled transcriptional silencers (tTS) that bind promoters responsive for rtTA in absence of the effector doxycycline (Dox). Addition of Dox prevents binding of tTS thus relieving repression, promotes binding of rtTA and thereby switches the promoter from an actively repressed to an activated state. 



Results

Of several tTS – fusions between a modified Tet repressor and transcriptional silencing domains – tTSKid was found to be most effective in reducing the activity of two target promoters. Ten to 200 fold repression is seen in transient expression whereas in stably transfected HeLa cells the regulatory range of the rtTA system was increased by three orders of magnitude.



Conclusions

The new system appears particularly suited for the transfer of toxic genes into appropriate chromosomal sites as well as for tight regulation of genes carried by viral or episomal vectors. Copyright © 1999 John Wiley & Sons, Ltd.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/%28SICI%291521-2254%28199901/02%291%3A1%3C4%3A%3AAID-JGM4%3E3.0.CO%3B2-Y

A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells

Inducible gene expression systems for higher eukaryotic cells.

Publisher Summary This chapter describes the successful use of the tetracycline-inducible expression system for the analysis of dynamin's function in receptor-mediated endocytosis and offers some practical advice for its establishment. Others have also used this system to establish stable cell lines inducibly expressing, for example, cyclins that regulate progression through the cell cycle. Earlier, detailed biochemical and morphological analysis of dynamin's function in the process of endocytic-coated vesicle formation required the generation of stable transformants. As endocytosis is important for cell viability, it was expected that the generation of stable transformants expressing mutant dynamin would require the use of an inducible expression system. Given these constraints, an optimal inducible system would allow stringent control of the expression of individual genes and enable a rapid and complete on/off switch of gene activity. Various inducible eukaryotic promoters responsive to heat shock, heavy metal ions, or hormones have been described. However, these were less desirable because of uncontrollable leakiness of the inactivated state and/or pleiotropic effects caused by the inducers themselves.

Sabine Freundlieb

Papers

Transcriptional activation by tetracyclines in mammalian cells

Co-regulation of two gene activities by tetracycline via a bidirectional promoter

A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells

Inducible gene expression systems for higher eukaryotic cells.

Tightly regulated and inducible expression of dominant interfering dynamin mutant in stably transformed HeLa cells.