Mysterious, Magical Milk

It's paper time!!! I told you I was leading up to something.  And here it is!
This paper
"Changes in Proteasome Structure and Function Caused by HAMLET in Tumor Cells" was published in PLOSone April 2009.  It's free, open access so go and read it!  Or if you want, read on and I'll try to take you through it.

So we established that HAMLET is a variation of the alpha*-lactalbumin protein.  HAMLET is formed when alpha-lactalbumin (encoded by the LALBA gene, remember that one?) is partially unfolded, loosing a Ca2+, and hooking around a fatty acid called oleic acid.  This can occur in acidic environments, like say, in the stomach.  Let's recap that: a major protein found in human breast milk changes to a cancer-fighting molecule when it passes through the stomach.  In a clinical study, topical HAMLET killed off skin cancer cells.  In humans with bladder cancer, an infusion of HAMLET caused tumor cells to be shed and tumors shrank. Interesting.

So this team, coming from the Lund University in Sweeden and the A*STAR group from Singapore, wanted to look at how HAMLET might be killing tumor cells. First they showed in cell culture that HAMLET selectively kills off several type of cancer cells but not normal differentiated cells.  Also, alpha-lactalbumin does not kill either type of cell. They also showed through fluorescent-labeled HAMLET that 50x more HAMLET gets inside tumor cells than alpha-lactalbumin.  So, somehow HAMLET is selectively entering and killing tumor cells, but not normal cells.

Next they turned to the proteasome.  They showed in vitro (not in cells, in cell lysate and chemistry experiments) that HAMLET binds to the 20S proteasome.  They also did co-immunoprecipitation and used antibodies specific to HAMLET to pull it out of a cell slurry, and they showed that particles of the 20S proteasome were attached to HAMLET.  Finally they used immunofluorescence in cultured tumor cells to show that HAMLET and the 20S proteasome were located very close to one another.  These data are not water-tight but are suggestive that HAMLET and the proteasome are interacting.

So, HAMLET interacts with the proteasome and also selectively picks off the tumor cells.  What could it be doing?  The group next showed that HAMLET is resistant to proteasomal degradation, by showing it is resistant to the enzymes trypsin and chymotrypsin, and it also is a partial proteasome inhibitor, by showing proteasomes from HAMLET treated tumor cells don't degrade other proteins.  Using mass spectrometry, they see some changes to subunits of the proteasome in HAMLET treated tumor cells: namely, structural subunits, a nuclear localization signal, and some catalytic subunits.  This suggests HAMLET gums up the normal workings of the proteasome.  They also show this on protein immunoblot; proteasome subunits from HAMLET treated tumor cells end up on a different part of the gel, suggesting the size or composition of those subunits is altered. Finally, they show increased fluorescent staining of proteasomes in HAMLET treated tumor cells, again suggesting the trash can is broken.

That's it.

So where does this get us?  It does appear that HAMLET has an effect on the normal workings of the proteasome.  It gums up the trash can, and the authors speculate this creates a toxic effect on the rapidly growing tumor cells.  Normally, gumming up the trash compactors of the cells cause disease, but this is suggesting mucking with the garbage disposal can be beneficial.

Why tumor cells and not normal cells?  Well, they didn't do any of their proteasome studies in normal cells, and they only looked at differentiated normal cells.  Would it have an effect on proliferating normal cells?

And what is this doing coming from breast milk?  It's great that HAMLET has anti-tumor effects, but what is it doing for babies?  Is it just a random by-product or is it somehow offering early protection for our babes in the early years?  There exist a few anecdotal stories out there of women providing breast milk to cancer-striken loved ones, and they loved ones recover.  Just anecdote, or did the conversion of alpha-lactalbumin to HAMLET help these cancer patients?

Related - does it have to be HAMLET from human milk, or does bovine alpha-lactalbumin converted to HAMLET do the same thing?

Well, just thought that was a pretty little study. As a nursing mom and a cancer researcher (on hiatus) it's things like this that fascinate me and make me glad I'm doing what I'm doing.


P.S.  Sorry this one was long-winded and kinda jargon-y.
P.P.S  You can email comments/questions/concerns/suggestions here!




* anyone know how to make the alpha symbol on the Mac using keyboard strokes?

Gene Wednesday: LALBA

Righto!  Here's post number six, and guess what.... six people signed up to follow this little blog. Talk about motivating factors.

Today is the inaugural Gene Wednesday.  I love genes.  I love genetics.  So here's where I'll spotlight one gene.  I promise, all these posts this week, though they seem disjointed, are leading to something.

Today's gene is LALBA. It codes for the protein human alpha-lactalbumin.  It's located on human chromosome 12q13 (the long arm of chromosome 12).  As you can probably guess by the name, it's the principle protein in milk.  But it's oh so much more.  It binds to to another protein, lactose synthase, and makes lactose, the principle sugar found in milk.  LALBA is only expressed in mammary tissue, and is hormonally regulated.  It is highly conserved among mammals, and mice that have both copies of LALBA deleted make a thick milk lacking lactose that does not sustain the pups, showing that LALBA is involved in lactose synthesis and that lactose is necessary for sustaining infants.



Alpha-lactalbumin appears to do much more than make lactose.  It can bind together into a multimeric protein that has apoptotic properties - it can help kill tumor-like cells but not normal cells in culture.   Some data suggest it also has microbicidal activity And recently, a protein dubbed HAMLET (human alpha-lactalbumin made lethal to tumor cells) is alpha-lactalbumin folded in a different way when exposed to acidic environments that, as the somewhat awkward name suggests, appears to specifically kill tumor cells. So one gene, one protein, lots of different functions.  Neat!

Term Tuesday: Proteasome

Today's term is Proteasome.  Never heard of it?  Cool.  Let's get started then.

 Like every good city, someone needs to take out the trash and the proteasome is the one to do it for a cell. The proteasome is the trash compactor and recycling center for proteins in a cell. The proteasome is a big, barrel shaped protein complex in a cell (we're talking eukaryotes here) that degrades and disassembles bad proteins.  By bad proteins, I mean proteins that were made or folded improperly, proteins that no longer are needed, and proteins that have been damaged, say by heat shock.

The proteasome is made up of several protein complexes that arrange themselves, I kid you not, like a kitchen trash can with a pop-up lid.  The details get my head spinning, so I'll probably not do justice to the amazing research behind discovering how it works.  But here it goes.  The main barrel of the proteasome trash can, called the 20S particle, is made of small proteins aggregating together to make rings. There's 7 rings in the barrel, and the outer two contain regulatory elements that can sense what proteins are entering to be degraded. The lid of the trash can, called the 19S regulatory cap, senses the proteins to be degraded and feeds them into the barrel.  It takes energy to destroy a protein and the energy comes in the form of using ATP, the main fuel in the cell.  The energy spent here is in the binding of the bad protein to the 19S and 20S particle.  There's yet another part of the trash can, the 11S particle, and it regulates degradation of short proteins or peptides.

Proteins are generally tagged for destruction by a small chemical modification called ubiquitin.  Attaching onto the amino acid Lysine, the ubiquitin is placed on there by yet another complex of proteins called ubiquitin ligase.  The ubiquitin ligase is the trash collector: it identifies the proteins in the cell that need to be recycled.  You guessed it, there's several proteins associated with ubiquitin ligase, and it's where the specificity comes into play. When a protein is tagged for destruction, it attaches to the 19S (or 11S if it's small) particle and is fed into the 20S particle.  It's here that it gets chewed into small peptide chains, which can be further degraded and recycled into new proteins.  Nice and tidy.

Proteasomal degradation of proteins plays a huge role in the normal control of cell division, homeostasis, and stress response.  However, sometimes the proteasome degradation process goes awry, and can lead to disease.  Too much proteasomal activity can lead, supposedly, to autoimmune disease.  Not enough can lead to things like Alzheimer's disease and cancer.

Remembering back to high school biology (quite a few years back, I won't tell you how many) I never learned about proteasomes. It's because they probably weren't identified yet as parts of the cell. They were only really discovered in the '80's.  And work that identified it was deemed worthy of the Nobel Prize in 2004.  Really, we all should celebrate those who know how to take out the trash.

P.S.  I'll get to actually putting in reference and attributions here soon.  I promise.

Method Monday: Cell Culture

I have my training in genetics, and more specifically cancer genetics.  So a great deal of my work has been with little cells growing rapidly on plastic, in pink-colored media, in a warm, humid incubator.  This is the world of cell culture, also called tissue culture. It's also the iconic image of a laboratory worker: someone in a white coat, gloves and goggles, laboring away with their arms in a plastic chamber and transferring reddish liquid from one flask to another.

Hint: most of those pictures are totally staged.  I never wore goggles.

Here's what cell culture is all about.

Cells are harvested from a source, usually a hunk of tissue, (we'll come back to that) and are gently teased apart both mechanically via scissors and chemically with enzymes, typically trypsin, collagenase, or dispase, that chew up the connections between the cells.  Then, the cells are soaked in a liquid media, and placed in a vessel, either a flask or a petri dish.  These are usually plastic treated with poly-L-Lysine, creating a positively charged surface to make the cells stick better.  Then the cells sit in an incubator set to body temperature (37C, or 98.6F) that is infused with carbon dioxide.  This keeps the cells at a happy pH. Then let the experiments begin!

What is in the media?  Well, it's got some salts, some sugar, some minerals.  Think of it as a really expensive form of Gatoraide.  Usually there's antibiotics too, because good cells and bad bacteria don't mix.  That can be it, and that is called minimal media - just enough to keep the cells alive.  But most cells need more than that to grow.  So extras are added, such as amino acids, the building blocks of proteins, and often serum.  Harvested from calves or fetal calves (don't ask, you don't want to know), serum a blood product chock-full of growth factors and goodies that help cells grow.  Oh, and the pink color?  It's phenol red, a dye that indicates the pH.  Cherry red is good, purple is bad.

So where do these cells come from? Pretty much any hunk of tissue will do, from human, mouse, dog, Chinese hamster, you name it.  For a lot of purposes, cell lines have already made, and can be purchased.  Very convenient, because it allows multiple researchers to use the same cells in their experiments and it's easier to compare data.  For example, one frequently used cell line is a human cervical cancer cell line called HeLa.  The name comes from "Henrietta Lacks."  The cells were made in 1951, and nearly every cancer lab since has used those cells. Hows that for immortality? Other times researchers have to make their own cell lines.

Some main categories of cells people use are primary, immortal, and stem cell lines.  Primary means they're straight from a living being, and typically have a short time they can be used due to senescence - a process of aging that limits the number of times a cell can divide. In one lab, I routinely made primary cell culture from mouse hippocampal neurons. Not cells that can easily live and divide for many generations.  Immortal cell lines have supposedly limitless replicative potential are those that have been pushed through senescence, through crisis, and live on. Or they have been induced to live on and on by introducing viral genes that allow them to keep on growing.  Both methods rely on changing the expression of genes that normally keep cells from dividing out of control.  And stem cell culture takes cells from the inner cell mass of an embryo which are then grown on a feeder layer of cells, with careful media conditions.  These cells are undifferentiated and supposedly have limitless replicative potential.

So, there's books and books written on cell culture, and really neat things researchers use it for.  I remember in one lab working on muscle differentiation, and I could take muscle stem cells, add a couple growth factors into it and a few days later, watch the differentiated muscle cells twitch in the dish.  Freaky. But other than playing around, cell culture gives us a way to poke and prod into the biology of cells in a way not otherwise possible in a living organism. Yes, there's drawbacks and limitations.  Cells in a dish do not behave like cells living in a body.  But for a vast amount of basic biology and early drug testing, cell culture is a key way to gain insights and knowledge.