Twenty more winks

In Episode One we alerted ourselves to the large amount of evidence saying that a good night’s sleep really is essential if you wish to reduce your chances of a wide variety of medical misfortunes. But what do we know about how molecules respond to sleep disruption to produce such nasty effects?

Molecular Clocks

Life on earth depends on energy sent forth by the sun and, in synchrony with the rotation of our planet, many of the inner workings of mammals fluctuate over each period of roughly 24 hours. This pattern is called the circadian clock, its most obvious manifestation being the sleep-wake cycle. Over the years considerable evidence has accumulated that the link between shift-work and cancer is probably due to circadian rhythm disruption and suppression of nocturnal production of a hormone called melatonin. All living things make melatonin (in mammals in the pineal gland of the brain) and it signals through a variety of protein receptors on cells to regulate the sleep-wake cycle but it also plays a role in protecting DNA from damage.

Melatonin production is regulated by the circadian oscillator, itself controlled by two sets of proteins that control each other’s expression in a feedback loop. Thus one pair, CLOCK and BMAL1, activates Cryptochrome and Period. They in turn repress CLOCK and BMAL1 – the upshot being that the activities of both pairs oscillate over a day-night cycle: as one goes up the other comes down. These central regulators are encoded by evolutionarily ancient genes (two for Cryptochromes and three for Period proteins). In plants and insects CRY1 responds to light but in mammals CRY1 and CRY2 work independently of light to inhibit BMAL1-CLOCK.

Two interlocked feedback loops control clock protein expression

CRY-CLOCK

OUTCOME: ≈ 24 hour cycle expression of PER & CRY

BMAL1 & CLOCK 12 hours out of phase

Alarming the Clock

So having sounded the alarm that just one night’s sleep shortage has obvious effects, what do the genes make of it? Well, the short answer is they get upset. A recent study took blood samples from a group of normal people and found that more than 700 genes (about 3% of our total number) significantly changed their level of expression over 1 week of insufficient sleep (5.7 h) by comparison with 1 week of sufficient sleep (8.5 h). About two-thirds were reduced whilst one-third was up-regulated (made more of their protein product). Unsurprisingly, among those that went down were the major clock regulators. It’s worth noting that the sleep perturbation in this experiment was relatively mild – intended to be similar to that experienced by many individuals. The genes most strongly affected play roles in a wide range of biological processes – DNA structure (hence gene expression), metabolism, stress responses and inflammation. The responses of genes to changes in sleep patterns are not the result of mutation (i.e. changes in the sequence of DNA)  but, at least in part, they’re caused by small changes in the structure of DNA. {These are epigenetic modifications – any modification of DNA, other than in the sequence of bases, that affects how an organism develops or functions. They’re brought about by tacking small chemical groups either on to some of the bases in DNA itself or on to the proteins (histones) that act like cotton reels around which DNA wraps itself}. Thus there is evidence for gene silencing by hyper-methylation of CRY2 (adding methyl groups (CH3) to its DNA) and the converse effect of hypo-methylation (removing methyl groups) of CLOCK occurs in women engaged in long-term shift work and is associated with an increased risk of breast cancer.

Inflaming the Problem

The cells that mediate inflammation and immune responses also have circadian clocks – meaning that normally these processes are rhythmically controlled and clock disruption (for example by sleep loss) affects this pattern. Disabling the clock in mice (by knocking out CRY altogether) switches on the release of pro-inflammatory messengers and knocking out one of the Period genes (PER2) makes mice cancer-prone – reflecting the fact that MYC (the key proliferation driver) is directly controlled by circadian regulators and is consistently elevated in the absence of PER2.

Clock Faces

The mass that comprises a tumour is a mixture of cells – cancer cells and normal cells attracted to the locale – so it’s a quite abnormal environment and in particular there may be regions where the supply of oxygen and nutrients is limited. This is sensed as a stress by the cells, one response being to lower protein production until normal conditions are restored. If this doesn’t happen within a given time the response switches to one leading to cell suicide. One way in which overall protein output can be reduced is by activating an enzyme (IRE1α) that breaks down code-carrying messenger RNAs that direct assembly of new proteins. Remarkably, it has emerged that one of the mRNAs targetted by IRE1α is the core circadian clock gene, PER1. The degradation of PER1 mRNA means that less PER1 protein is made, which in turn disrupts the clock. However, it seems that PER1 has other roles that include helping the cell suicide response – a major anti-cancer defence. All of which suggests that disruption of the IRE1α/ PER1 balance might have serious consequences. Indeed IRE1α mutations have been found in a variety of cancers including brain tumours in which low levels of PER1 are an indicator of poor prognosis. The IRE1α mechanism coincidentally activates the transcription factor XBP1 (as well as PER1 mRNA decay) and one target of XBP1 is the gene encoding a messenger (CXCL3) that makes blood vessels sprout offshoots. Thus this master regulator suppresses cell death, activates proliferation (lowering PER1 deregulates MYC) and promotes new blood vessel formation.

A Tip for Snoozing

If you’re still wide awake it just goes to prove the utter fascination of biology – but today’s story says that you have to find ways of, if not falling asleep, at least courting insensibility (as Christopher Fry put it). If it’s a real problem for you may I make a really radical suggestion? Turn to our physicist friends and select from their recent literary avalanche. A ‘brief history of …’ something or other will do fine. It’s a knock-out! Sweet dreams!!

References

Möller-Levet, C.S., Archer, S.N., Bucca, G., Laing, E.E., Slak, A., Kabiljo, R., Lo, J.C.Y., Santhi, N., von Schantz, M., Smith, C.P. and Dijk, D.-J. (2013). Effects of insufficient sleep on circadian rhythmicity and expression amplitude of the human blood transcriptome. PNAS 110, E1132-E1141.

Fu, L.N. et al. (2002). The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo. Cell 111, 41-50.

Zhu, Y. et al. (2011). Epigenetic impact of long-term shiftwork: pilot evidence from circadian genes and whole-genome methylation analysis. Chronobiol Int, 28, 852–861.

Pluquet, O. et al. (2013). Posttranscriptional Regulation of PER1 Underlies the Oncogenic Function of IREα. Cancer Res., 73, 4732-4743.

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Powdering Your Nose and Other Parts

If you were asked ‘What is the worst thing about being a research scientist?’ you might well come up with ‘Feeling stupid every day’ – especially if you’d read Martin Schwartz’s wonderfully funny and incisive essay ‘The importance of stupidity in scientific research’ pointing out that research means battling with the unknown. Bad though that is, I can tell you, on the basis of collecting absolutely no data whatsoever, that 100% of scientists would answer ‘Literature’ – or to be slightly more expansive ‘Keeping up with published research.’ To give the rest of mankind a feel for their problem, suppose you work on a gene called MYC which is one of the most powerful cancer drivers: the Web of Knowledge database lists 3,839 hits for MYC as a topic and 468 with it in the title (which means you really ought to read those papers!). So far this year! That’s six months  worth!!

Dusting down the literature

Broadly speaking, scientific literature comes in two categories: a huge one that you might call worthy but dull and a tiny one to which you ought to say ‘Wow!’, that is, there’s some amazing revelation about the way life works, a brilliantly clever method or some stunning insight. But there are two other small classes of which we rarely speak. One is, of course, stuff that is poor (or worse still plagiarized) and should never have been published. The other is perfectly OK – indeed you might even say ‘good someone’s done it’ – it’s just that your heart sinks when you see the title because you know what’s in store.

talc-powder

My latest heart-sinker is a zippy little thing called Genital powder use and risk of ovarian cancer: a pooled analysis of 8,525 cases and 9,859 controls and it has that effect because the title tells all. They’ve tackled a question that’s been around for 30 years, namely whether applying talcum powder to the nether regions can cause cancer of the ovaries, by pulling together data from separate studies with mixed conclusions, so that a kind of average emerged from the haze as a modest increased risk.’

What’s my problem?

Being certain that such a title will be picked up by the press and reported in a misleading and over-hyped fashion. Step forward the MailOnline (Women who regularly use talcum powder to keep fresh raise their risk of ovarian cancer by almost a quarter SHOCK HORROR!!). OK, I added the last two words but they were there by implication. It has to be admitted that the scientists didn’t help by calculating Odds Ratios (the ratio of the odds of an event occurring in one group to the odds of it occurring in another group), with the inevitable result that they were interpreted as ratios of risks, which overestimates the effect. However, if journalists actually bothered to read the papers they latch on to, it might occur to them that a balanced picture might be conveyed by quoting what the scientists themselves said. In this case the odds ratio was 1.24 which they summarized as ‘Genital powder use was associated with a modest increased risk of epithelial ovarian cancer.’ It would also help the non-scientist reader to put things into context by, in this case, noting that for ovarian cancer the average lifetime risk is about 1.4%. Thus even if you did have an increase of one quarter, the risk is still less than 1.8%.

The ordinary reader might also appreciate a comment on some of the problems faced by such studies. Not the least of these is that they are retrospective (i.e. they asked folk to recall what they used, when and how). It’s not difficult to be skeptical about the precision of the responses, especially when you’re tiptoeing around in what might be called delicate areas, and that’s before you mention the different wording in each study of questions that were pretty convoluted anyway. It’s also worth noting that the analysis showed no increase in risk with prolonged use, which is a little odd (recall that for smoking the more you do it the higher your chances of lung cancer).

Anything else worth adding?

Talcum powder, for this is what we’re talking about, is made from talc which is mostly magnesium, silicon and oxygen and the powder is, of course, widely used because it absorbs moisture and reduces friction, helping to keep skin dry and rash-free. Asbestos, another silicate, occurs together with talc in nature, and it causes the form of lung cancer called mesothelioma. Before 1976, talcum powder was commonly contaminated with asbestos but since the 1970s talcum products have been asbestos-free. There is evidence both in humans and rodents that talc particles can travel up through the genital tract and alight on the surface of the ovaries. Such particles can cause inflammation, one way in which cancer development can be set off, but there is no evidence that talc does promote ovarian cancer in this way.

Ideally in looking for cause and effect, scientists like to get a handle on mechanism. Somewhat surprisingly, for an effect that is modest at most, there is the glimmering of a lead. It comes in the form of a family of enzymes that can detoxify carcinogens (they’re glutathione S-transferases) but the genes encoding two of them, GSTM1 and GSTT1, are missing in about 50% and 20% of Caucasians respectively – so, of course, their activity is lost. There is one study showing that women with GSTM1-present and GSTT1-missing have a stronger association between talc use and ovarian cancer risk. The number of cases is small and it is possible that the effect is not real. It’s also not at all clear how the actions of this combo might interact with the effects of talc. Nevertheless, it is striking that it’s the only pairing of these two genes that shows an association.

What’s a girl to do?

1. Don’t read anything by a journalist that talks about Odds Ratios because the odds are they won’t have a clue what they’re on about.

2. Do read Thou Shalt Not Report Odds Ratios’, Mark Liberman’s witty but brutal evisceration of two ‘science editors’, Mark Henderson of the London Times and Steve Connor of the Independent newspaper.

3. Note that the authors of this study say that genital powder exposure is associated with a ‘small-to-moderate increased risk.’ – nothing stronger than that.

4. Remember that there’s no evidence that talcum powder applied anywhere other than the genital area can cause any problems and that includes the lungs. Even rats forced to inhale talc for 6 hours a day, five days a week for over two years were reluctant to get lung cancer although the incidence did increase in females (maybe they were just trying to escape the Dickensian smog ‘Strewth guvnor, I ’ardly get to see the nippers these days: may as well end it all by getting lung cancer’).

5. Bear in mind that the International Agency for Research on Cancer (IARC) classifies talc-based body powder as a class 2b carcinogen “possibly carcinogenic to human beings.”

6. Be aware that the major factors increasing the risk of ovarian cancer are (1) increasing age, (2) family history of breast or ovarian cancer, (3) being overweight and (4) hormone replacement therapy, whilst having children and breastfeeding them as well as taking the pill reduce the risk.

7. Finally, if the possibility of a slight increase in a small risk really spooks you, avoid orifice powdering and let nature take care of things. Or, if you’re really desperate for friction-free movement, use cornstarch powder: it’s a carbohydrate and there’s absolutely no evidence that it is a risk factor for ovarian cancer.

References

Terry, K.L., Karageorgi, S., Shvetsov, Y.B. et al. (2013). Genital powder use and risk of ovarian cancer: a pooled analysis of 8,525 cases and 9,859 controls. Cancer Prevention Research Published OnlineFirst June 12, 2013.

http://www.dailymail.co.uk/health/article-2343974/Women-regularly-use-talcum-powder-increase-risk-ovarian-cancer-24.html

Gates, M.A., Tworoger, S.S., Terry, K.L. et al. (2008). Talc use, variants of the GSTM1, GSTT1, and NAT2 genes, and risk of epithelial ovarian cancer. Cancer Epidemiology Biomarkers & Prevention 17, 2436-2444.

http://itre.cis.upenn.edu/~myl/languagelog/archives/004767.html

Delay Olympics for eight years, says biochemist

No he didn’t because that would just be silly wouldn’t it? What my colleague Chris Cooper from the University of Essex was reported as saying by The Independent was “Delay awarding London 2012 Olympic medals for eight years” because he thinks it will take that long for drug tests to separate who was playing the game (cricket, obviously) from the cheats – the word taken from Chris’s book Run Swim, Throw, Cheat.

The current front-runner in the game of Beat the Biochemists appears to be erythropoietin (EPO) – a natural hormone that makes us produce more red blood cells. That’s handy if you go in for endurance events (like surviving t.v. coverage of the Olympics). The Boffins went 1 – 0 up recently by coming up with a test that picks up EPO after it’s been injected.

You don’t know how lucky you are!

In the second leg the Scoundrels have hit back with fiendish cunning. A key factor that regulates whether we make EPO is oxygen availability. Lower oxygen means more red cells needed. But for that to happen there has to be a molecular messenger that can sense oxygen levels. There is: it’s a protein called hypoxia-inducible factor (HIF, of course) that under normal conditions gets broken down very quickly – by a process that needs oxygen. So when oxygen drops HIF lasts longer, makes more EPO and that makes more red cells. The crafty bit is finding another molecule that stabilizes HIF – in effect, enables it to survive even when there’s plenty of oxygen. HIF stabilizers are potentially important in treating some diseases and they’re just the ticket if you want to cheat in the 5,000 km bog snorkel.

There’s a bit of a concern because HIFs play an important role in helping cancers to grow so, adding that to the stress of wondering if you’re going to be nicked, it’s all going to be a bit of a strain for any ‘athletes’ who succumb to temptation. But there’s a time-honoured way of dealing with stress and this isn’t the moment to spoil the ship. A pack a day should do the trick.

Sorted. It’s all systems go for gold in the true Olympian spirit, Lucky Strikes in one pocket, HIF stabilizers in the other, morals in the changing room. The Boffins are scuppered, at least until they can find a way of detecting the invisible EPO driver, unless of course the fags give things away. What the score-line will be when we hear the merciful blast of the final whistle on 12th August is anyone’s guess – but for once I wouldn’t bet on the Boffins.

So who was the idiot responsible for the title of this piece? What could have possessed him? I have no idea but here’s a guess. What if he thought: let’s ban the Olympics for two rounds – and come 2020 everyone will say “Gee, what a great eight years we’ve had with none of the colossal waste of money on these staggeringly over-hyped, extraordinarily tedious and somewhat malodorous events. Let’s not bother any more.” Give that man a medal – without delay!

Reference

http://www.independent.co.uk/sport/olympics/news/delay-awarding-london-2012-olympic-medals-for-eight-years-says-biochemist-7917937.html