[If you cannot see the movie, you can watch it here.]

It was quite interesting, wasn’t it? Let me summarize the main points raised in the talk. Eva suggests that cancer might simply be part of a natural response mechanism aimed at repairing tissue damage. Somehow most of our body, according to Eva, has not had enough time to evolve to handle the response perfectly, so when the damage is prolonged, cancer often arises. However, there are tissues in our body, such as skeletal muscle, which are well-adapted to sustaining damage of various kinds, and is therefore able to keep cancer under control by means of either moderating nutrient access to this cancer, or by inducing differentiation of cancer cells in situ. Eva suggests that one day we might be able to use the mechanisms behind the repair system (basically, cancer) to repair tissue AND control the spread of cancerous tissue. In this sense, cancer could almost become a cure, a form of “therapy”, as she calls it.

I am not to extensively criticize her point of view, as there are some obvious issues with it. There seems to be an underlying assumption that cancer stem cells are always normal stem cells recruited to a damaged region of the body for repair purposes, and that only after getting there these cells become cancerous. This is a mistake, from my point of view. It makes perfect sense that there is an accelerated regeneration process in a damaged tissue. Think of the skin: if you cut yourself, now your skin stem cells will have to work harder to produce new cells, so to repair the damage. I am also aware that prolonged inflammation states associated with tissue damaged have been showed to predispose to cancer.

But much cancer does not necessarily happen in response to inflammation states. Lung cancer is a good candidate for the inflammation militia, but what about leukemia? Brain cancer? Breast cancer? It is known that many cancers are caused by a minuscule population of stem-cell-like cells that cause the disease: some of these probably were originally normal stem cells, but there is mounting evidence that at least in some cases, cells that were not stem cells acquire, through a mix of somatic insults and genetic predisposition, a new undifferentiated state – and that “un-differentiation” and proliferation are actually caused by separate mechanisms.

The talk often oversimplifies, but what really struck me is the idea that, indeed, it is very rare to hear of skeletal muscle cancer. In fact, this must be the first time in my life I even hear of the concept! It never crossed my mind, for a moment, how interesting this could get. Eva suggests that, somehow, skeletal muscle tissue can regulate angiogenesis to limit tumor growth. But I think the explanation she offers last is the one that makes more sense: there must be strong differentiation factors, maybe myoD itself, that are limiting tumor growth – so that, even if the original nucleus of stem cells remains, it can only rarely grow to a full-blown metastasis, as all other progenitors rapidly differentiate into muscle cells.

The idea is fascinating, and easily explorable. All you need is test it in immuno-deficient mice using tumor cells from syngeneic mice bearing a visible/otherwise detectable marker. You can look at metastatic frequencies in various tissues and, if really the frequency is significantly lower (and metastases smaller) in skeletal muscle, you could run some FACS and look at the proportion of cancer progenitor cells in the micrometastases versus the normal ones – if her idea is right, you would expect to see very few of them, as well as many differentiated cells originally derived from the tumor.

What intrigues me about all this is manly that the cancer cells are not different in one or two things from the other cells. Their entire behavior is changed – and only some form of powerful “undifferentiating agent” could do that. In cancer research, we often try to look for the “magic molecule”. But I think it is time to start thinking in terms of a system of interactions involving DNA, epigenetic modifications, proteins and the relations (pathways) between them. The whole system defines cellular behavior, and it is this behavior, not just a couple of molecular players, that is actually changed in some key cells when cancer arises.

Thus, although I am skeptical that cancer might be a therapy, the focus on differentiation and changes in cellular behavior are thing to keep working on, as well as watching carefully, in the next years. Especially for those who are working on stem cell therapies – whose most common side effect is…cancer.

Post Scriptum: I have been included in the latest edition of Gene Genie. The main topic of this edition was the inauguration of Google Health, but you will find a lot more related to health in general, and genetics specifically, by reading the Genie hosted over at Highlight Health.

To do this, the authors extracted DNA from three century-old pouch young stored in alcohol, and a dried pelt. The extraction methods including classic ethanol precipitations and sucrose gradients (to eliminate contaminants). As usual in this cases, the DNA resulted to be fragmented, but the researchers were aided in their effort to verify their results by the presence of different DNA sources, and the choice of a well-characterized sequence, the transcriptional enhancer of a collagen gene (Col2a1). Comparing the results obtained from PCR reactions run on DNA extracted from the three different sources helped assure that the DNA was not contaminated (as human collagen genes would be similar, but not identical, to those belonging to the Tasmanian tigers).

The researchers then decided to study the function of this element (reconstituted by PCR) in vivo. Multiple copies of the enhancers were added in front of the he human β-globin basal promoter fused to lacZ and followed by a polyadenylation signal – therefore producing a chimeric reporter gene to use in their assays. This reporter would reveal the expression pattern of the genetic element, as well as the strength of expression in developing tissues. This reporter construct was then injecting in murine zygotic pronuclei, and its expression assayed at different stages of mouse development.

As you can see from the third figure in the paper, you can see that the reporter is most strongly expressed in regions where cartilage is forming – the skull, the tail, and the developing limbs. In section g, you can even distinguish the digits in the developing mouse forelimb. These results indicate that, regardless of some variation in the strength of the expression, the transgene made out of the “extinct DNA” is expressed in the same pattern as that if the endogenous transgene – which is also expressed at sites of cartilage development and growth. This therefore means that the function of the modern murine element, and the one belonging to the extinct Tasmanian tiger, is actually conserved. It also suggests that this method could be used to analyze the function of genomic material extracted from extinct specimens in vivo.

While this does not mean that we will be able to resuscitate the Tasmanian tiger any time soon (if not at all), it gives us another glimpse on the practical power of evolutionary theory, and the incredible genetic conservation among species, even those that evolved quite separately from each other.

Pask, A.J., Behringer, R.R., Renfree, M.B., Svensson, E.I. (2008). Resurrection of DNA Function In Vivo from an Extinct Genome. PLoS ONE, 3(5), e2240. DOI: 10.1371/journal.pone.0002240

The situation for food can coating resins (also known to release biologically active amounts of BPA) is similar, with industry studies contrasting research on the amount of leaching from food cans.

Does this mean that one of the sides is lying? Well, a honest skeptical answer would be…no. There is no need for conspiracy theories to explain these disagreements. Mostly, the debate is fueled by the fact that nobody really knows how to assess the effects of xenoestrogen in humans and their significance. What amount of BPA do we need to ingest such that it will have an impact on our health? Should we consider amounts that are ingested all at once, in a short period of time, or over a lifetime? Is the concept of “lifetime xenoestrogen load” useful or not?

Answering these questions is what would unlock the current debates, as by now all sides are at least willing to accept that BPA has unquestionable biological activity as a xenoestrogen. Here is a short list of papers (there are many more) discussing the biological effects of BPA absorption in animals, effects directly related to estrogenic activity:

• Prepubertal exposure to compounds that increase prolactin secretion in the male rat: effects on the adult prostate. Biol Reprod. 1999 Dec;61(6):1636-43 (PMID: 10570013)
  
• The xenoestrogen bisphenol A induces growth, differentiation, and c-fos gene expression in the female reproductive tract. Endocrinology. 1998 Jun;139(6):2741-7 (PMID: 9607780)
• The environmental estrogen bisphenol A stimulates prolactin release in vitro and in vivo. Endocrinology. 1997 May;138(5):1780-6 (PMID: 9112368)
• The developmental toxicity of bisphenol A in rats and mice. Fundam Appl Toxicol. 1987 May;8(4):571-82 (PMID: 3609543)
• Bisphenol A in the aquatic environment and its endocrine-disruptive effects on aquatic organisms. Crit Rev Toxicol. 2007;37(7):607-25 (PMID: 17674214)

You might have noticed that these papers are about ten years old. What is the current consensus on the biological activity of BPA, and its mode of action in animals? Here are a few more recent papers addressing these questions:

• In vivo effects of bisphenol A in laboratory rodent studies. Reprod Toxicol. 2007 Aug-Sep;24(2):199-224. (PMID: 17683900)
• An evaluation of evidence for the carcinogenic activity of bisphenol A. Reprod Toxicol. 2007 Aug-Sep;24(2):240-52. (PMID: 17706921)
• In vitro molecular mechanisms of bisphenol A action. Reprod Toxicol. 2007 Aug-Sep;24(2):178-98. (PMID: 17628395)

The second paper is of special interest, as it summarizes the results of a panel discussion organized by the National Institutes of Health (NIEHS, NIDCR) and the United States Environmental Protection Agency, which “convened an expert panel of scientists with experience in the field of environmental endocrine disruptors, particularly with knowledge and research on bisphenol A (BPA)”. This review suggests that there is a wide scientific consensus regarding the possible role of BPA in carcinogenesis. The consensus arising from the panel discussions is succintly summarized in the Conclusions section of the paper:

Based on existing evidence, we are confident of the following:
1. Natural estradiol-17β is a carcinogen as classified by the International Agency for Research on Cancer [37], [106] and [107].
2. BPA acts as an endocrine disruptor with some estrogenic properties among other hormonal activities.

Based on existing evidence, we believe the following to be likely but requiring more evidence:
1. BPA may be associated with increased cancers of the hematopoietic system and significant increases in interstitial-cell tumors of the testes.
2. BPA alters microtubule function and can induce aneuploidy in some cells and tissues.
3. Early life exposure to BPA may induce or predispose to pre-neoplastic lesions of the mammary gland and prostate gland in adult life.
4. Pre-natal exposure to diverse and environmentally relevant doses of BPA alters mammary gland development in mice, increasing endpoints that are considered markers of breast cancer risk in humans.

Based on existing evidence, the following are possible:
1. BPA may induce in vitro cellular transformation.
2. In advanced prostate cancers with androgen receptor mutations, BPA may promote tumor progression and reduce time to recurrence.

The paper also highlights areas of BPA research that require further enquiry:

1. Does BPA exposure induce or promote cancers in mammary and prostate? What is its mode of action?
2. Does BPA increase cancer susceptibility in estrogen-target organs (prostate, mammary gland, uterus, vagina, testis, ovary, etc.)?
3. Does BPA reprogram target tissues during development through epigenetic mechanisms, including epigenetic marking of genes and morphogenetic processes involving tissue interactions?
4. What are the most appropriate life stages for examining BPA-induced cancer susceptibility?
5. Under what conditions might BPA promote DNA and/or microtubule aberrations?
6. Identify biological consequence of long term, low-dose exposure on genomic integrity, cooperation with oncogenic insult and tumor management.
7. Development of carcinogenesis paradigms with relevance to humans for assessing the ability of BPA to alter cancer risk.
8. What species/strains are the most appropriate for assessing BPA-induced cancer susceptibility?
9. Developing three-dimensional culture models to assess the mechanisms involved in altered morphogenesis of the target organs that may lead to neoplastic development.
10. Epidemiology studies and development of new methodologies to evaluate BPA-cancer risks in humans.
11. Development of markers for total xenoestrogen insult in humans.

Do you remember the recent studies published in the journal Cancer Research, which received strong media attention? One showed that BPA induces changes in gene expression in breast cancer cell lines coherent with those of high-grade lesions; the other suggested that BPA is also able to alter the epigenetic profile in the progeny of BPA-treated epithelial cells. These papers are already addressing points 1 and 3 in the “further research needed” list, even if partially.

Past and present data, as well as the current consensus, seem to suggest that it might be prudent to limit BPA intake in humans until further research determines whether BPA is relatively safe – as we already know that it is not absolutely safe. Until the time when more rigorous studies are conducted, and larger data sets on humans collected, we will not know for sure whether BPA poses a significant danger to human health – it is left to the individual, but also to public health agencies around the world to make a decision on whether to forbid BPA use especially in places where it can enter the food chain…or not. But it seems that there is reasonable evidence that BPA release in the environment should be limited as much as possible, as smaller organisms are sensitive to much smaller amounts of BPA than humans seem to be.

Making decision in relation to BPA is made even more complicated by the fact that there are many estrogen-like compounds in our environment which are already in the food chain, and which we can absorb by consuming both animal and vegetable products: BPA absorption might only be the tip of the iceberg when it comes to xenoestrogen intake. It would be useful to see what the “total xenoestrogen insult” is in an average adult who consumes meat, vegetables and dairy, and to see what role BPA is playing to increase this insult. Only then we will be able to assess whether cancer risk arising from BPA ingestion is significant, or whether we would do better to worry about different sources of xenoestrogen.

KERI, R., HO, S., HUNT, P., KNUDSEN, K., SOTO, A., PRINS, G. (2007). An evaluation of evidence for the carcinogenic activity of bisphenol A. Reproductive Toxicology, 24(2), 240-252. DOI: 10.1016/j.reprotox.2007.06.008

I am therefore going to split this article into two parts. In this first part, I will introduce the concept of xenoestrogens, talk a little bit about what bisphenol A is and what is used for, and tell the story of the discovery of its – initially unsuspected – effects on systems usually regulated by estrogen and similar hormones. In the second part, I will talk about the research regarding BPA potential toxicity, carcinogenicity, and the medical and ecological implications of BPA presence in our environment.

First of all, let’s get to know the steroid hormones, a hormone family estrogens are happy members of. That’s right – estrogens: this is a sub-family of steroid hormones. There are three main estrogens in humans: estradiol, estriol, and estrone, produced from androgens. The process of producing steroid hormones (both male and female) from cholesterol is called steroidogenesis. Here is a sketch of the process (click on the thumbnail to get to the full-sized image).

Estrogens are important for the determination of female secondary sexual characters, and not only in humans. They function during development, and during certain types of tumorigenesis. On the other side of the family, we find the androgens, the male-determining hormones. They are also derived from cholesterol…and in fact, estrogens are produced after chemical modification of some androgens. A commonly known androgen is testosterone.

Some man-made (say, BPA) or natural (for instance phytoestrogen) compounds with a structure close to that of the steroid hormones are able to sometimes reproduce the physiological effects of these hormones. These are sometimes called xenoestrogens – literally, foreign estrogens. How was it discovered that BPA is in fact a xenoestrogen?

One of the studies documenting this discovery was a perfect example of serendipity. Researchers at Stanford University were trying to find out whether the yeast Saccharomyces cerevisiae is able to produce estrogen. To do this, they decided to grow the yeast in common flasks, and to run an assay for endogenously produced estrogen. They quickly realized that radioactively-labelled estradiol added to the growth medium was being displaced by estrogen coming from another source. This source turned out to be not the yeast, but the polycarbonate flasks themselves. The substance causing the effect was purified using high-performance liquid chromatography (HPLC), and was found to be BPA. Tests on mammalian cells showed that BPA was effectively an estrogenic compound, able to induce expression of progesterone receptors, and to bind estrogen receptors in mammalian cells.

We now know that BPA’s ability to mimic endogenous estrogen has the ability to affect the reproductive system, and sexual development – at least in frogs. The sex ratio in frogs being exposed to BPA is altered, and feminization can be observed when frogs are exposed, as tadpoles, to varying concentrations of BPA – and this correlates with an increased expression of the estrogen receptor.

What is BPA, and why is it used? Bisphenol A is a compound that can be used to create polymeric plastics. Most specifically, BPA finds its main use in the production of polycarbonate plastics, which are hard to shatter, and therefore used, among other things, in laboratory glassware, bottles for drinking water and food containers. Polycarbonates are also used as sealers – found in the lining of plastic bottles or tin cans. Polycarbonate plastics are extremely resistant to heat and mechanical stress, but bleach and strong alkali can cause the release of BPA from the plastic. And apparently, these plastics initially leak low levels of BPA when exposed to boiling water, about 55 times more than when the water is at room temperature.

But is this leakage dangerous for human and animal health? Is the evidence in favor (or against) its health effects reliable, unbiased and coherent? Is it safe to use polycarbonate containers for your water and food? We will find out more about this in the next post.

Post Scriptum: this post was kindly included in the latest edition of Tangled Bank.

Citations

Krishnan, A.V. (1993). Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving.. Endocrinology, 132(6), 2279-2286.

Levy
, G. (2004). Bisphenol A induces feminization in Xenopus laevis tadpoles. Environmental Research, 94(1), 102-111. DOI: 10.1016/S0013-9351(03)00086-0

LE, H., CARLSON, E., CHUA, J., BELCHER, S. (2007). Bisphenol A is released from polycarbonate drinking bottles and mimics the neurotoxic actions of estrogen in developing cerebellar neurons. Toxicology Letters DOI: 10.1016/j.toxlet.2007.11.001

Maragou, N., Makri, A., Lampi, E., Thomaidis, N., Koupparis, M. (2008). Migration of bisphenol A from polycarbonate baby bottles under real use conditions. Food Additives & Contaminants, 25(3), 373-383. DOI: 10.1080/02652030701509998

These studies on “fruitless” flies are some of the coolest studies on gender-specific behavior around. Although the BBC suggestively calls this a “mind-control sex swap“, this is definitely not a sex swap. One characteristic of fruitless flies is that their sexual characteristics are perfectly normal – the males are fertile, and so are the females, and there is no deformation of their bodies. So we are really not looking at the sex of the flies – but rather, at their gender. Which is what makes these studies so intriguing.

First of all, let me briefly explain how courtship works in Drosophila melanogaster. The male is the only one that can be said to display courtship behavior: when a male intends to court a female, it uses wing vibrations to produce a “courtship song”. This song can be identified because of the specific frequencies of the sounds emitted by the male. There are two “modes” of emission, known as the sine song, at ∼140–170 Hz, and the pulse song, constituted of brief and repetitive amplitude modulations in a range of ∼150–300 Hz. Females respond to this by allowing the male to copulate (or not; whether the male is successful depends on a variety of other factors, including winning “fights” with other males).

What about these fruitless (fru) flies? It was already known that fruitless encodes a gene product necessary for the determination of sex-specific courtship behavior in flies, and that the products of this gene exist in two main versions produced by alternative splicing: a male version and a female version. A previous study done in Barry Dickson’s lab showed that the male version is necessary for the determination of male courtship behavior and sexual orientation. The gene is in fact also able to induce male courtship behavior in females when spliced in a male-specific manner (causing the male version of the protein to be produced in females). On the other hand, loss of male-specific splicing in males causes the disappearance of all typical male courtship behavior.

This whole situation is complicated by the fact that there are several different alleles (variants) of the fru gene, able to alter not only courtship, but sexual orientation as well. For example, males with certain mutant forms of fru display an enhanced form of homosexual behavior. What do I mean by that? You might want to see for yourself in this video from one of the Dickson papers. Click on the image to go to the video, which gives a new dimension to the phrase “love train”.

Now we can get back to today’s study. Researchers working at Yale and Oxford have decided to look at the neuronal circuitry expressing the products of the fru gene in male and female flies, and to see what would happen when one activates this neuronal circuit in fru mutants of both sexes. The experiments showed that activating this circuit results in different responses in males and females, and that the responses are dictated by the version of the fru protein expressed by the animal. Thus, wild-type males and male-fru-expressing females can produce the wing movements and the courtship song; wild-type females will move their wings and produce a sound, but not a courtship song.

The neuronal circuitry was artificially activated by shining light on the neurons, which were made to express a light-sensitive ion channel.

What does this mean? When ions are allowed into neurons by ion channels, the neurons depolarize – and the depolarization wave moves all along the membrane of the neuron, till it reaches the synapse, where neurotransmitter release occurs. All you need to to do activate a neuron in a controlled way is to make it express an ion channel you can control. Ion channels need energy in the form of ATP to work, and therefore providing ATP can induce depolarization. You can then use a form of ATP linked to another compound: this compound makes the ATP “inactive”, but when you shine light on it, the “normal” ATP is released, and the ions channels open.

How do you make sure that the channels are present in the neurons you are studying? You use a promoter specific to those neurons – so that the gene coding for the channels makes protein only there. And what better promoter to study fru-expressing neurons…than the fru promoter? In fact, that is the one used in this study.

This short video summarizes the results of the study. I love when the narrator says that “to make a female sing like a male, all you need to do is to turn on one gene, and chop her head off”. It does give the wrong impression, doesn’t it? The head is chopped so that the female does not stop “singing”, as that behavior usually requires interaction with another fly, and it is intermittent. Which is not helpful if you want to analyze the song of that one fly in the experiment.

Now, apart from the results and the hype, you might still be wondering what are the implications of the study, from a more neurobiological point of view. What the results suggest is that behavioral differences between the sexes might not be necessarily due to differences in neural circuitry, but in the presence or absence of sex-specific regulators of such circuitry. Although it is usually true that there are differences in the overall neuronal structure of males and females (with males usually having extra neurons dedicated to male-specific behavior, which is something that is surely true of the nematode C. elegans), the fact that some shared circuitry might also be part to fundamental sex-specific behaviors is also something that needs to be considered.

As a last note, in case you are wondering: while some sources (which do not attach any citation to this statement) say that the fruitless gene is not present in mammals, I have run my own search, and it turns out that humans might have a homologue of the fly gene. The name of the homologue is ZBTB22 (which tells me this gene has not been well characterized), a transcription factor, just like fruitless is. Does this mean that it has the same function in humans? Well, given that we do not make courtship songs using wings, it probably doesn’t.

Curious facts: we have known about gay fly males for quite a while. In fact, the fru mutants were initially created because of X-ray radiation in 1963, and there is a 1989 paper describing the effects of the fruitless defects in males, and tracking them back to a chromosomal inversion, which must have probably been caused by DNA breaks and repairs following radiation exposure in the original strain. Nowadays people are looking at different mutated alleles of fru, not at the original version created through radiation exposure.

Citation

Clyne, D., Miesenböck, G. (2008). Sex-Specific Control and Tuning of the Pattern Generator for Courtship Song in Drosophila. Cell, 133(?), 354-363.

Male fly image courtesy of Wikipedia Commons