Download or read book Mammalian Sam-checkpoint in Altered Cancer Metabolism written by Da-Wei Lin and published by . This book was released on 2014 with total page 117 pages. Available in PDF, EPUB and Kindle. Book excerpt: The majority of my work was focused on the regulation of a mammalian cell cycle checkpoint that is controlled by the metabolite S-adenosylmethionine (SAM). Proper cell growth and proliferation require sufficient nutrient supply to synthesize cellular building blocks such as nucleic acids, proteins, and lipids. Yet, every single cell is challenged daily by environmental stress such as redox hazards, DNA damage, and metabolic stress. In order to maintain DNA integrity and faithfully transmit genetic information, mammalian cells have evolved a plethora of cell cycle checkpoints to ensure cell cycle arrest when conditions are not suitable for cell division. S-adenosylmethionine (SAM) is a key metabolite required to faithfully duplicate epigenetic marks during DNA replication. I am interested in how mammalian cells monitor intracellular SAM levels and halt proliferation when SAM levels are inadequate for growth. My discoveries in this field can be divided into two parts: (1) SAM limitation induces p38 activation and triggers cell cycle arrest in the G1 phase. (2) Characterization of the cell cycle response in breast cancer cells during methionine stress, which leads to reduction of intracellular SAM levels. In addition to the work on the SAM-controlled cell cycle checkpoint, I also developed a high throughput screening platform based on yeast cells for identification of small molecules that can reactivate p53 mutants found in human cancers. I discovered that reducing intracellular SAM levels through methionine free culturing conditions, methionine adenosyltransferase (MAT) inhibitors, or targeting of MAT with shRNAs results in a robust G1 cell cycle arrest with decreased Cdk2 activity concomitantly with lowered Cyclin E levels. Surprisingly, the activity of the main cellular nutrient sensor mTORC1 was unchanged throughout SAM-checkpoint activation. Instead, the mitogen-activated protein kinase p38 and its downstream checkpoint kinase MK2 are responsible for SAM-checkpoint activation and induction of cell cycle arrest in the G1 phase. Another metabolic stress sensor, the tumor suppressor protein p53 was unaffected during the first phase of SAM limitation. Instead, p53 level was increased at a much later time point after G1 arrest had already been established. p53 most likely functions in the maintenance rather than the induction of SAM-checkpoint. In contrast, p38 and MK2 were crucial for the initiation of the SAM-checkpoint and their inhibition allowed cells to progress into S-phase despite of suboptimal intracellular SAM levels. Importantly, a substantial amount of sub-G1 cells were discovered when the SAM-checkpoint was overridden by p38 or MK inhibition, reaffirming that the SAM-checkpoint guards cell integrity. Lastly, I discovered that that p38 inhibition and SAM limitation induce cell death synergistically, which highlights a pharmaceutical potential of this novel metabolic checkpoint. The SAM-checkpoint is closely linked with the phenomenon known as "methionine dependency of cancer cells" or "Methionine/ Homocysteine-effect (Met/ Hcy- effect)", which describes a phenotype in transformed cells where they stop proliferation if methionine is replaced with its direct metabolic precursor homocysteine in the growth medium (Met-Hcy+ media). Anchorage independent MB468 breast cancer cells are strictly dependent on methionine. When these cells were place in Met-Hcy+ media, a robust S-phase block was induced with low Cdk2 activity but unchanged Cyclin E level. In contrast, anchorage dependent isogenic cell pair MB468Rev cells were able to maintain proliferation in Met-Hcy+ media. Because all organisms synthesize SAM from methionine, it is likely that SAM levels were affected when methionine was replaced with homocysteine. This unique metabolic phenotype in anchorage independent breast cancer cells is likely due to reduced flux through the Hcy-Met-SAM axis. However, the underlying links between the transformed phenotype and Met/ Hcy-effect needs further investigation. I also contributed to establish a high throughput screening platform in yeast that aims to identify compounds that reactivate p53 mutants found in human cancer. I engineered a yeast strain that will only survive and proliferate in uracil free media in the presence of p53 activity. The system I developed allows multiplexed screening formats through mixing p53-tester yeast strains expressing different p53 cancer mutants in one well. A set of ten p53 cancer mutants representing the entire spectrum of p53 mutants found in human tumors were selected for the multiplex screen format and used for pilot screening. In addition, I engineered a secondary screen into the same yeast strains using the p53-dependent Mdm2 promoter with a LacZ reporter gene. This secondary screen provides a quantitative readout and helps to exclude false positive hits. Altered metabolic pathways in cancer cells hold our hopes for next generation cancer therapeutics. Uncovering the molecular components for SAM-checkpoint activation is just the first step of the upcoming discoveries that will contain many attractive drug targets. It is certain that with advancing technology and additional work in this area, we will soon acquire the knowledge that would enable us to target cancer cells according to their specialized metabolic status that ultimately bring personalized medicine to each and every one of the cancer patients.