MTT is 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Cancer and cell biology research concepts
What education/training after high school does it take to become a (life) scientist? General Path What I Did• Bachelors degree (~4yrs) in a science • Bachelors degree (in Finance) but as part of a individually developed curriculum took enough biology and 2000-2004• (Only sometimes) Masters degree 1- chemistry to get 2 minors 2yrs, classes & perform focused research project• PhD degree, some classes and full-time • PhD in Molecular Carcinogenesis research (have to discover something at the UTHSC/MDACC, Smithville 2004-2011 NEW!), usually paid for RA/TA, 5-7+yrs, TX write a 100-200 page dissertation at end• Postdoctoral fellowship(s) 2-5+ yrs – full • TRIUMPH postdoctoral fellow, at time research, learning how to write the University of Texas MD 2011-now grants, more research training, how to Anderson Cancer Center, Houston run a lab etc TX
Education in pictures Newark DESmithville (1hr from Austin), TX Houston, TX
Cancer – the disease which affects nearly 2M Americans, and causes >500K deaths/year• What is cancer? • Cells that acquire mutations in their DNA that result in abnormal growth – divide continually when they are not supposed to, and don’t die when they receive death signals.• Tumors form when cancer cells divide and form a mass.• Tumors grow their own blood vessels to get nutrients to survive (angiogenesis)• Soon cancer cells become invasive – take over the normal tissue, and spread around the body making metastases (e.g. in the lungs, or brain or bones). This is why people die from cancer – usually primary tumors do not kill people. Cancer biologists like to compare cancer cells and normal cells to understand what went wrong, and how to kill the cancer cells alone!
Tools of the trade – examples of different breast cancer cells taken from human patients growing on plastic plates in lab • Cells can be cultured from humans, mice, other rodents, insects etc…. • We can culture cancer cells indefinitely in solutions of glucose and amino acids (to make proteins)!
Proteins function in pathways in cells Challenge is there are 100s of these pathways & they are all interconnected in ways we don’t fully understand! Some proteins have different functions depending on WHERE they are and what proteins they interact with!
PhD Thesis: “ATM signaling to TSC2: Mechanisms and Implications for Cancer Therapy”Key questions:• How do cells (including cancer cells) detect and respond to DNA and oxidative damage?• Are these protein pathways linked to cell survival or death?• Can we target these survival pathways to improve cancer therapy (chemotherapy, radiation, big surgeries)?Methods overview:• Study protein localization in the cell and their function (for example whether they are active or not) using fluorescence microscopy and cell fractionation• Used genetic approaches to study whether a process depends on a single protein – used cells that either lack the protein, or used a technique called RNA interference to reduce the amount of the protein I was interested in.• Used drugs to inhibit pathways – such as protein export from the nucleus
Examples of techniques used in a cell biology lab Western blotting – we make protein mixtures from cells or animal tissues, and run gels to separate the proteins based on size. Using antibodies to proteins we can measure amounts of these proteins and their modifications.We can also break up cells and use selective solutions of chemicals (buffers) to only get cytoplasm, membranes or nucleus
Supplementary Figure S3 Techniques in a cell biology lab (cont.) a HEK 293 cells b HEK 293 IgG WT RQ RW RG IgG WT RQ PEX5 (light) Flag-TSC2 PEX5 (dark) IP: FlagFluorescence and confocal microscopy – use this to observe Flag-TSC2 PEX5 protein localization (what organelles are important?) c e Flag PMP70 Merge/DAPI HEK WT IgG WT RQ Flag-TSC2 Myc-TSC1 Flag PMP70 Merge/DAPI Myc-TSC1 RQ Flag-TSC2 HeLa cells Flag PMP70 Merge/DAPI RG Flag PMP70 Merge/DAPI RW d Flag Calnexin Merge/DAPI RQ ells
More cell biology techniquesElectron microscopy – observe structural Animal/human tissue studies information inside cells without using – immunohistochemistry antibodies to detect individual proteins staining for proteins using antibodies then chemical reactions that generate brown color
Major findings of my PhD work (team project)• Key discovery – a very important protein (called ATM) plays different functions depending on where it is localized in the cell • Before my work, most people thought it mainly functioned to sense DNA damage in the nucleus, and recruit other proteins to REPAIR the damage or trigger cell death if too much damage. • I showed convincingly that a separate pool of this protein that didn’t have to leave the nucleus could respond to a oxidative damage and trigger autophagy • Autophagy is a recycling process cells use to degrade damaged organelles and certain proteins • Often used by cancer cells to survive stresses such as chemotherapy and radiation. • Understanding how and when this applies can help us design ways to block cancer cell survival.
Cancer and the cell cycle • Cell cycle deregulation is virtually universal feature in cancer • Cancer cells both lose the “brakes” (eg Rb and p53 tumor suppressor genes) & often have a “stuck gas pedal” Key question – how can we target this defect to selectively kill cancer cells? Once we do it in cells and mice, can we design a human clinical trial…
Some more common techniques relevant to cell biology I use now Doing high-throughput drug screening MTT measures mitochondrialfor good drugs and combinations using reducing activity in the cell – is MTT a surrogate for live cell number LIVE CELLS Yellow Purple insoluble powder chemical (in H2O) Increasing dose of drug
Studying the cell cycle: What phase of the cell cycle does a particular drug affect? Flow cytometry – uses lasers to Cells are incubated with a fluorescent dye (propidium capture information about single iodide) that binds to DNA then the machine measures cells, size, shape, fluorescence etc how much fluorescence per cellBasic Principle: Amount of DNA S G1 G2/M (DNA synthesis)1X DNA Between 1- 2X DNAcontent 2X DNA content content
Observations about how cells dieOur old friend, electron microscopy Autophagy Apoptosis Mitotic Necrosis catastrophe If we know by what method(s) cells die, we can try to understand why!
More cell death – all about the nuclei!Nuclear morphology as a measure ofapoptosis (one way cells can die) –cells are stained with a fluorescent bluedye (called DAPI) TISSUE NO TISSUE DAMAGE USUALLY DAMAGE /INFLAMMATORY NO TISSUE RESPONSE DAMAGE
Questions?Email me @ firstname.lastname@example.org or talk to me on twitter @thecancergeek