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Innovative measurement of cellular stress allows development of new cancer drugs

DNA damage in our cells can induce cell death or potentially cancer-causing mutations. However, cells have developed sophisticated repair mechanisms that rapidly begin to remedy such damage in order to prevent long-term consequences. One of these repair mechanisms is the attachment of poly(ADP-ribose) (PAR) to proteins. To investigate this process in more detail, Dr. Aswin Mangerich and colleagues from the University of Konstanz and the MIT have developed a bioanalytical platform based on isotope dilution mass spectrometry to quantify PAR levels with high specificity and sensitivity. The results provide the researchers with new insights into the cellular PAR metabolism, which is useful for the development of anti-cancer and other drugs.

Dr. Aswin Mangerich (centre), Dr. Rita Martello (left) and Prof. Dr. Alexander Bürkle (right) from the University of Konstanz and their colleagues from the Massachusetts Institute of Technology (Cambridge, USA) have developed an innovative measurement method that allows the exact quantification of poly(ADP-ribose). © University of Konstanz

Poly(ADP-ribosy)lation is the post-translational modification of proteins similar to the phosphorylation or acetylation of proteins, amongst other modification processes. Poly(ADP-ribosyl)ation is mediated by poly(ADP-ribose) polymerases (PARPs) which add PAR, a biopolymer with chemical similarities to nucleic acids, to proteins. PARPs are involved in a number of cellular repair processes where DNA damage occurs as a result of natural metabolic processes or external effects in the form of radiation, heat or chemical substances.

“It is assumed that PARPs are involved in the recruitment of cellular DNA repair tools to the damage site, thereby supporting and coordinating a range of different cellular repair mechanisms,” explains Dr. Aswin Mangerich, a molecular toxicologist from the University of Konstanz who is specifically interested in the PAR metabolism. Poly(ADP-ribosyl)ation can affect proteins’ enzymatic activity, cellular localisation and interaction with other proteins. 

In addition to DNA repair, poly(ADP-ribosyl)ation is also involved in other physiological and pathophysiological cellular processes, including the transcription of genes, the regulation of cellular inflammatory responses and programmed cell death. In addition, anti-cancer drugs based on a variety of pharmacological poly(ADP-ribosyl)ation inhibitors (so-called PARP inhibitors) that have been shown to enhance the effect of established tumour therapies are currently in clinical development. Certain anti-cancer agents damage the DNA of the affected cancer cells, resulting in their death. “PARP inhibitors have been shown to have a direct tumour-inhibiting effect (following the principle of synthetic lethality) in tumours with a specific genetic constellation, for example hereditary breast cancer caused by BRCA gene mutations,” Dr. Mangerich adds. 

More than the tip of the iceberg

The cellular poly(ADP-ribose) metabolism is a highly dynamic process as the quantity of cellular PAR can either increase a hundredfold after DNA damage has occurred, or decrease as a result of the rapid degradation of PAR by cellular enzymes. This dynamic and variability makes it quite difficult to precisely determine the amount of PAR. Current measuring methods are based on the indirect detection of PAR using antibodies that bind to PAR. However, this approach only has limited sensitivity and a limited linear measurement range, which renders the detection of physiological PAR levels and quantification rather difficult, if not completely impossible. “We wanted to develop a new method that would allow us to precisely determine poly(ADP-ribose) levels in cells and tissues with the aim of exploring the physiological and pathophysiological role of this type of post-translational modification,” Dr. Mangerich says.

The new method is based on the direct detection of poly(ADP-ribose) using a mass spectrometer: poly(ADP-ribose) is initially extracted from cells and tissues using biochemical methods, then enzymatically cleaved into smaller fragments of a characteristic mass, which are separated using HPLC and detected in a mass spectrometer. PAR quantity is determined with the help of an internal standard that is added to the sample during extraction. Isotope-labelled poly(ADP-ribose) is used as internal standard, the chemical-enzymatic synthesis of which was specifically developed for use with this new method. The isotope-labelled internal standard has the same biochemical characteristics as cellular poly(ADP-ribose), but has a different mass due to the incorporated isotopes and can thus be unequivocally identified in a mass spectrometer. “We are thus able to compensate for the technical variations that might have occurred during sample preparation,” Aswin Mangerich explains.

The method can also be used for the direct estimation of the number of poly(ADP-ribose) molecules. “The situation can be compared to an iceberg: the approach that uses antibodies to detect the PAR molecules only detects the tip of the iceberg, while the mass spectrometric approach also detects the iceberg mass underwater,” said Dr. Mangerich explaining the advantages of the new method. The mass spectrometric approach also enables the structural characterisation of PAR molecules, which provides the researchers with information about biopolymer length and degree of branching. This knowledge is crucial for being able to explore certain biological questions at the molecular level.

Development of new chemotherapeutic drugs based on PAR measurement

Steps in the newly developed method for quantifying poly(ADP-ribose) (PAR). Left: Schematic of all work steps involved in the quantitative measurement of PAR in human blood cells (PBMC = peripheral blood mononunclear cell, e.g. lymphocytes and monocytes). Right: biochemical and bioanalytical principle underlying the mass spectrometric quantitation of PAR. The quantitative cellular extraction and enzymatic digestion of PAR leads to molecules that are specific for different structures of the PAR molecule and that can be analysed with a mass spectrometer. © Martello, R., Mangerich, A., Sass, S., Dedon, P.C., Bürkle, A., 2013. Quantification of cellular poly(ADP-ribosyl)ation by stable isotope dilution mass spectrometry reveals tissue- and drug-dependent stress response dynamics. ACS Chemical Biology

The new method has already led to new findings. “We have been able to show that the poly(ADP-ribosyl)ation reaction varies between different cell types and individuals, which is useful information for our understanding of cancer development and in the treatment of cancer,” says Dr. Mangerich highlighting the results.

The method opens up completely new possibilities for investigating poly(ADP-ribosyl)ation reactions. “Physiological poly(ADP-ribosyl)ation reactions are a largely unexplored area,” Dr. Mangerich says. Poly(ADP-ribosyl)ation can also play a major role in drug development. The new method can be used to pharmacologically characterise the mode of action and efficacy of chemical PARP inhibitors that are currently undergoing clinical development for the treatment of tumours. For example, it is possible to check whether the inhibitor under investigation actually exerts an inhibitory effect in the target tissue.

“Moreover, poly(ADP-ribosyl)ation activity has been shown to be an excellent marker for the response of tumours to certain cytotoxic drugs,” says Aswin Mangerich. The exact characterisation of the poly(ADP-ribose) metabolism in tumour tissue can therefore provide information about the likelihood of success of certain tumour therapies. 

Further information:
Dr. Aswin Mangerich
Molecular Toxicology 
Faculty of Biology 
University of Konstanz
E-mail: aswin.mangerich(at)uni-konstanz.de

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/innovative-measurement-of-cellular-stress-allows-development-of-new-cancer-drugs/