Cutting Edge Cancer Diagnosis: Circulating Tumor Cells

This year’s AACR annual meeting focused a great deal on the vindication of a very old idea:  “seed and soil” model of metastasis.  The idea that little seed cells from a tumor circulate through the body looking for fertile ground to implant into and grow a metastatic tumor.  It’s only very recently that the technology has existed to provide a mechanism to detect these circulating seed cells.  This knowledge has proven to be very important in predicting which patients will respond to different therapies.

This video is taken from the only FDA approved technology to detect these circulating tumor cells (CTCs).  For any of you interested in what I do for a living, this is the clinical outcome of some technologies that I worked on at the “research use only” non-clinical level.  Being able to tag cells with magnetic particles or beads and separate or enrich from that binding.

It’s five minutes long and includes a commercial in the middle, but I thought it was a cool “infographic” format.  Very CSI!

Understanding the Hayflick Limit, part 2

Telomerase makes new DNA from an RNA template

Last time, we started into telomeres, and I hinted at the function of telomerase.  Telomerase is an enzyme found in most organisms that adds bases to the ends of your chromosomes, extending the “aglet” (telomere) of the chromosome.  It does this in a rather startling way… it has its own RNA sequence that it uses as a template for making new DNA.  This is startling, at least to a virologist, in that it’s very much like what a retrovirus does.  Telomerase is considered a reverse transcriptase, meaning it takes an RNA template and makes complementary DNA from it.  This is opposite from the usual flow of information in the cell  (DNA –> RNA –> protein).  In the case of telomerase, what is copied is a simple motif of a a few bases repeated many thousands of times in a row.

Normal embryonic cells have active telomerase, as do some adult cells that have to divide often, like white blood cells.  Most cells, though, have their telomerase gene “shut off” so that no more can be made.  Remember that every time a cell divides, those telomeres get shorter by a few hundred bases, eventually getting too short to hold the ends, and the chromosome becomes unstable.  There are some diseases that result from premature shortening of the telomeres (Werner syndrome), and the patients have signs of premature aging (“progeria”).

The idea that the upper limit of our lifespan (“cellular senescence”) is somehow tied to a single biological phenomenon is intriguing.  If you, dear reader, immediately begin wondering whether this ticking clock can be silenced, or turned back, you are not alone.  I imagine every young scientist first learning about telomeres and telomerase imagines themselves as the saviors of mankind, or perhaps fulfilling the lines of the “Necronomicon”:

Cthulhu: telomerase positive

That is not dead which can eternal lie.
And with strange aeons even death may die.

So why can’t we just “turn on” the telomerase activity and stop the inevitable shortening of telomeres which causes cellular replication to fail in the elderly?  It has to be pointed out that some organisms do not succumb to senescence, and they are essentially immortal, although none of them are smart enough to clean up on long term, low risk investments.  Key examples are lobsters, jellyfish and coral.  Lobsters in particular are interesting because they have active telomerase throughout their lifespan, and they actually appear to be more fit the older they get.  That subject is a riddle that, so far as I know, remains unsolved.

Why do we poor mammals have to be content with a scant 100 or so years of life?  Why can’t we, like the lobsters, live forever?  The answer lies in one word:  cancer.  Cancer is built in to our biology.  Any system that requires exquisitely tight controls will eventually fail.  If that system governs the rate at which cells grow and divide, you will eventually have out of control growth.  The longer you walk this tight rope, exposed to mutagens in the air, the soil and the water, the greater the risk of failure.  Telomere shortening may have evolved as a way of keeping cancer in check, eliminating older cells that have been exposed to DNA damaging conditions.  Turn telomerase back on, and those geriatric cell lines will continue to accumulate damage, increasing with each division the chance of cancer developing.  It may not be a coincidence that the immortal animals live in dark oceans where UV damage is minimized.

Cruz Hernandez: 128 yrs old at death

That’s the cellular evolutionary view of senescence.  The organismal evolutionary view is more intuitive.  If old animals never die, and the number of animals that can be sustained in an ecosystem never changes, then the larger, older animals would outcompete the young, smaller animals for food and resources.  With no new generations, the gene pool becomes very static and the diversity remains low, making the population susceptible to pathogens.

“Futurists” like to talk about when the first immortal human will be born.  But I agree with Dr. Hayflick on this:

“When it becomes possible to slow, stop, or reverse the aging process in the simpler molecules that compose inanimate objects, such as machines, then that prospect may become tenable for the complex molecules that compose life forms. “

The concept of an immortal human with active telomerase ignores the central fact that older cells develop cancer.  Any proposal to immortalize humans must be prepared to deal with the escalating cancer and disease risk.  Aging and senescence are not just a matter of allowing cells to continue replicating.  It is dealing with the inborn errors of metabolism and biology.  As Dr. Hayflick suggests, if we can’t stop simple machines from breaking down, what hope do we have to prevent the same process in the very complex machine that is a human?

In a future post, I hope to explore those rare human cells that have achieved immortality as transformed cultures.

Understanding the “Hayflick Limit”

Leonard Hayflick, 1961 at the Wistar Institute

I met with an eminent scientist in the course of my work, and as part of the background research, I checked out his CV (curriculum vitae).  His background is substantial, Harvard education and NIH post-doc, but his mentor during his post-doc was Leonard Hayflick.  I knew about the Hayflick Limit from grad school, but I had never read the history of the phenomenon.  I thought it might make an interesting example of how science is conducted.  We’ll come to a definition shortly.

You may be aware that scientists routinely grow human cells in flasks.  It’s called cell culture or tissue culture.  We’re not talking about an arm or an ear, here.  The cells are only visible as a thin layer of translucent material, in the case of cells that adhere to a solid surface, or a slightly grainy slurry, in the case of cells in suspension.  We feed these cells, and they are bathed in, a solution containing salts, sugars, amino acids and other essential nutrients.  They also sometimes get antibiotics and growth stimulants.  Most also receive a healthy dose of serum, extracted from cow blood, in order to provide specialized proteins that stimulate growth.  We might say, for example, that our K-562 cells (immortalized cells from a patient with myelogenous leukemia) grow in RPMI1640 (a specific sugar/salt/amino acid solution) supplemented with 10% FBS (fetal bovine serum) plus pen/strep (penicillin/streptomycin) and L-glutamine (needed by some cells, and somewhat unstable in

Cell culture flasks with culture medium

storage).  It looks complicated, but is not much different from a recipe for chicken soup.  The flasks containing cells in their growth medium are placed in incubators, usually at 37 C and 5% CO2.  Periodically, the medium has to be changed, and if the cells are rapidly growing, they have to be passaged or “split”, removing a fraction of the growing cells, to avoid overgrowth.  We guard carefully against introducing infectious agents, since the cells have no immune system and any bacteria or fungus landing in the cell culture medium, loaded as it is with the materials of life, will rapidly overgrow, acidify the solution, and kill the cells.

So those are the very basics of growing mammalian cells in culture.  Now we come to the interesting part.  If I take a small scraping of your cheek and transfer them to a flask, they will grow for some time, then eventually they’ll stop growing and instead begin dissolving.  The number of divisions that occur before this happens is called the Hayflick limit.  Initially scientists assumed this was a technical problem with how they were culturing… not enough nutrients, or the introduction of bacteria or fungus pathogens… something was ending the life of the cells.

Some cells, however, could continue to grow almost indefinitely, almost always cancer cells or viral infected cells.

If they split the cells into two flasks, both cultures stopped replicating at about the same time, which made simultaneous infections unlikely.

Cells taken from embryonic tissues lasted much longer than mature cells taken from older patients.  Hayflick measured the number of times an embryonic cell could divide.  It converged on a value of about 50 divisions.  This would later be called the Hayflick limit.

Many cancer cells (~80%) do not obey the Hayflick limit, they are "immortalized".

So once the phenomenon was known and the puzzle was well-characterized, a number of hypotheses were advanced to explain the anomalous observation.  They focused on the exceptions to the Hayflick limit rules, cells that never stop growing, which we call immortalized cells.  These cells were almost universally taken from either cells infected with specific viruses, or from very active cancer cells.

So what do these two conditions have in common?  Both had damage to their DNA in certain specific regions that mediated cell division and self-destruct mechanisms.  Most cells recognize their genetic damage as a good reason to self-destruct, to undergo apoptosis.  These cells no longer had the ability to respond to that self-destruct signal.  That alone wouldn’t have been enough, though.  They had also acquired an enzyme that was able to add new sequences to the ends of their chromosomes.   These sequences are called telomeres.

Telomeres are to the chromosome as the aglet (the little plastic or metal collar) is to the shoelace.  The telomere prevents the DNA strand from “unraveling” by providing a long string of very tightly bound base pairs.  One of the consequences of copying your DNA is that a little chunk from the very end of each of your 46 chromosomes is lost every time they are copied.  When you are a fetus, you have nice long telomeres securing your chromosome ends.  As you age, as the result of replicating your DNA, they get shorter.  Eventually, they become too short to hold the ends together, and we think that’s why some cells must stop replicating.

Enter telomerase, a cellular enzyme that extends the length again.  In my next post, I’ll examine what it does in the cell; whether it can be harnessed to make us essentially immortal, and we’ll examine some of the claims of futurists and anti-aging researchers.  We’ll also look at why evolution favors our aging and eventual death, and those rare human genomes that have achieved near immortality.

I’ve just use something I learned on “Phineas and Ferb” in a science posting. Day complete.