A couple of important tumor suppressor genes have figured heavily in my past writings and in anti-aging science discussions, P21 and P53.  Another tumor suppressor gene may now be coming onto center stage, NRG1.  

Paying attention to the general press 10 days ago, one would get the impression that the NRG1 gene was just discovered and represents a breakthrough in cancer genetics with headlines such as Breast cancer gene discovery ‘most important for 20 years, Gene discovery is the biggest cancer success in 20 years, and Scientists find gene that stops some cancers in their tracks.  “Scientists have found a faulty gene linked to half of all breast cancers which experts have hailed as the most important discovery in the disease since the 1970s(ref).”    

 Actually, the NRG1 gene has been known for some time and its role in cancers has been investigated since 1998.  A Google search on the gene, Neuroregulin 1 (NRG1), returns 112,000 entries.  It is hardly newly-discovered.  The evidence for the tumor-suppressing properties of NRG1 is not new either.  It has been steadily accumulating and NRG1’s exact role in many cancers is still not well understood. 

The NRG1 gene encodes the protein Neuregulin 1 which is also known as NRG1.    “It is known that an extraordinary variety of different isoforms are produced from the NRG1 gene by alternative splicing. These isoforms include heregulins (HRGs), glial growth factors (GGFs) and sensory and motor neuron-derived factor (SMDF)(ref).” “The neuregulins are receptor tyrosine kinase ligands that play a critical role in the development of the heart, nervous system, and breast(ref).”  “NRG1 encodes growth factors that bind to tyrosine kinases ErbB3 and ErbB4, and can both stimulate cell proliferation and apoptosis. NRG1 is also quite frequently broken by chromosome translocations(ref).”  Among its other activities, it has been thought that “Neuregulin 1 (NRG1) is a leading schizophrenia susceptibility gene(ref),” and it may be involved in bipolar disorders(ref).

The October 2009 publication that set off the recent flurry of press reports is The NRG1 gene is frequently silenced by methylation in breast cancers and is a strong candidate for the 8p tumour suppressor gene.  “We found that most breast cancer cell lines had reduced or undetectable expression of NRG1. This included cell lines that had translocation breaks in the gene. Similarly, expression in cancers was generally comparable to or less than that in various normal breast samples. Many non-expressing cell lines had extensive methylation of the CpG island at the principal transcription start site at exon 2 of NRG1. Expression was reactivated by demethylation. Many tumours also showed methylation, whereas normal mammary epithelial fragments had none. Lower NRG1 expression correlated with higher methylation. — The short arm of chromosome 8 is frequently lost in epithelial cancers, and NRG1 is the most centromeric gene that is always affected. NRG1 may therefore be the major tumour suppressor gene postulated to be on 8p: it is in the correct location, is antiproliferative and is silenced in many breast cancers.” 

I touched on the role of DNA methylation in my blog post  DNA methylation, personalized medicine and longevity  and in Epigenetics, epigenomics and aging  where I pointed out “Already, certain DNA methylation changes are known to be associated with aging and others associated with certain diseases like lupus and scleroderma.”    I subsequently added a candidate theory of aging to my Anti-Aging Firewalls treatise EPIGENOMIC CHANGES IN DNA METHYLATION AND HISTONE ACTYLATION.  What is important in the current discussion is that in many cancer lines the expression of the NRG1 gene is shut down by methylation, meaning that no NRG1 tumor-suppressor protein is produced.  

The recent publication includes several laboratory displays graphically relating NRG1 expression to methylation in breast cancer and normal cells and concludes “– we suggest that NRG1 may be the principal tumour suppressor gene that leads to the loss of 8p in many breast and other epithelial cancers. NRG1 expression seems to be silenced in most breast cancers compared with the main types of mammary epithelial cell—this could be because tumours arise from a specialized population in which NRG1 is normally silenced, but we prefer the interpretation that NRG1 is silenced by aberrant methylation or other—as yet unknown—events such as promoter mutation. Expression of NRG1 in mammary cells is antiproliferative to the cells that express it, and array-CGH identifies NRG1 as the gene most likely to be a principal 8p tumour suppressor.” 

Actually the central results of this study were communicated in a poster presentation with the same title in May 2008.”  The story was not picked up by the press until more than a year later.

It is interesting to look at the history of discovery of the tumor suppressing properties of NRG1.  I mention only a few of the many publications which are building blocks for the most-recent  study.  The first relevant publication article I have seen cited was a 1998 one Neu differentiation factor (NDF), a dominant oncogene, causes apoptosis in vitro and in vivo.  “– we find that tumors induced by NDF (NRG1) display extensive apoptosis in vivo. NDF is therefore an oncogene whose deregulation can induce transformation as well as apoptosis.”

The 2000 study Chromosome translocations in breast cancer with breakpoints at 8p12 studied breakpoints and translocations on chromosome 8p in several cancer lines, relevant to the later discovery that these breaks and translocations were in NRG1.  This is one of several prior and subsequent studies looking at chromosomal rearrangements on 8p in cancers(ref).  One 2000 study states “We conclude that chromosome 8p carries a tumour suppressor gene or genes, the loss of which results in growth advantage of breast tumour cells, especially in carriers of the BRCA2 999del5 mutation.”  The recent research strongly suggests that that gene is NRG1.

The discovery of the involvement of two key genes in breast cancer, BRCA1 and BRCA2 was very exciting but led to a hunt for additional genes that might be involved, such as pointed out in the 2002 review article Genes other than BRCA1 and BRCA2 involved in breast cancer susceptibility.

The 2004 publication A Recurrent Chromosome Breakpoint in Breast Cancer at the NRG1/Neuregulin 1/Heregulin Gene states “We previously reported that five breast cancer cell lines have chromosome translocations that break in the NRG1 gene and that could cause abnormal NRG1 expression. — Breaks in NRG1 were detected in 6% (19 of 323) of breast cancers and in some lung and ovarian cancers. In an unselected series of 213 cases with follow-up, breast cancers where the break was detected tended to be high-grade (65% grade III compared with 28% of negative cases). They were, like breast tumors in general, mainly ErbB2 low (11 of 13 were low) and estrogen receptor positive (11 of 13 positive).

The 2005 paper NRG1 gene rearrangements in clinical breast cancer: identification of an adjacent novel amplicon associated with poor prognosis did some applicable legwork looking at rearrangements of the NRG1 gene and how they are specifically implicated in breast carcinoma oncogenesis.

The 2005 study Activation of ErbB2 by Overexpression or by Transmembrane Neuregulin Results in Differential Signaling and Sensitivity to Herceptin looks at the use of herceptin as a cancer treatment targeting NRG1.  “Treatment with the anti-ErbB2 receptor antibody Herceptin had an inhibitory effect on proliferation only in cells expressing neuregulin but not on cells overexpressing ErbB2, and its inhibitory activity was accompanied by a decrease in p21. These results suggest that Herceptin may also be of help in the treatment of tumors in which neuregulin feeds the tumoral tissue.”

A 2006 poster publication The NEUREGULIN1 gene and breast cancer is interesting in that it telegraphs the essence of the heralded 2009 results.  “Our current work shows that NRG1 expression is silenced in many breast cancer cell lines (17 out of 23 lines), as compared with normal breast cell lines. Western blotting experiments also indicate that NRG1 is downregulated at the protein level. To investigate whether NRG1 maybe repressed by epigenetic mechanisms, we examined DNA methylation at a CpG island present in the promoter and the first exon of the gene using bisulphite sequencing. This region is heavily methylated in 76.5% (13/17) of breast cancer cell lines that have no NRG1 expression. In contrast, the region is relatively unmethylated in normal breast lines, and in cancer cell lines expressing NRG1. Treatment of cancer cell lines with 5-aza-2-deoxycytidine, which abolished DNA methylation, activated the expression of NRG1 by 7–100 times. — These results suggest that DNA methylation is a key mechanism that silences NRG1 expression in breast cancer cells, and our current view is that NRG1 could be the long-sought tumour suppressor on 8p, with the translocations either inactivating the gene or producing aberrant transcripts.” 

Paul Edwards, a molecular biologist at Cambridge University, and his team were involved in this 2006 as well as the 2008 and 2009 reporting.  And there are a number of other studies over the years related to NRG1 and cancers.  A point I am making is that “breakthrough genetic discoveries” reported in the popular press are often the results of years of hard study by many people.  I do believe the latest results by Edward’s team represents solid and important progress.  However, we still only partially understand the role of NRG1 in other cancers and the likely role of NRG1 as a key tumor suppressor gene must be confirmed.   Despite sensational reporting, progress is achieved through many small incremental steps and there is far yet to go.

I am partial to an occasional slice of pizza despite my health-driven dietary compulsions. I love to sprinkle generous amounts of oregano on the slices and have often wondered what the health properties of that pungent herb are.  I recently learned that oregano is rich in rosmarinic acid and therefore, as my grandmother used to say “it is good for you.”  I review some of the things the literature says about rosmarinic acid here.

 “Rosmarinic acid, C₁₈H₁₆O₈, is a natural polyphenol antioxidant carboxylic acid found in many Lamiaceae herbs used commonly as culinary herbs such as lemon balm, rosemary, oregano, sage, thyme and peppermint^[1] (ref).”  It has anti-inflammatory, anti-bacterial, anti-viral, radio-protective, photo-protective, anti-allergenic and anti-oxidant properties.  It is somewhat structurally related to curcumin and resveratrol and is an ester of caffeic acid.  (See the blog entry Phytochemicals – focus on caffeic acid.)

Rosmarinic acid induces apoptosis in some cancer cell lines.  The 2009 paper Salvia Fruticosa, Salvia Officinalis, and Rosmarinic Acid Induce Apoptosis and Inhibit Proliferation of Human Colorectal Cell Lines: The Role in MAPK/ERK Pathway states “Aromatic plants of the genus Salvia (sage) have been attributed many medicinal properties, which include anticancer activity. In the present study, the antiproliferative and proapoptotic effects of water extracts of Salvia fruticosa (SF) and Salvia officinalis (SO) and of their main phenolic compound rosmarinic acid (RA) were evaluated in two human colon carcinoma-derived cell lines, HCT15 and CO115, which have different mutations in the MAPK/ERK and PI3K/Akt signaling pathways. These pathways are commonly altered in CRC, leading to increased proliferation and inhibition of apoptosis. Our results show that SF, SO, and RA induce apoptosis in both cell lines, whereas cell proliferation was inhibited by the two sage extracts only in HCT15. SO, SF, and RA inhibited ERK phosphorylation in HCT15 and had no effects on Akt phosphorylation in CO115 cells. The activity of sage extracts seems to be due, at least in part, to the inhibition of MAPK/ERK pathway.”

Rosmarinic acid has anti-oxidative activity.  Several publications point to this effect such as the 2008 paper Antioxidant activities of rosemary (Rosmarinus Officinalis L.) extract, blackseed (Nigella sativa L.) essential oil, carnosic acid, rosmarinic acid and sesamol.   The 2007 study Water and methanolic extracts of Salvia officinalis protect HepG2 cells from t-BHP induced oxidative damage states  “The most abundant phenolic compounds present in the extracts were rosmarinic acid and luteolin-7-glucoside. Both extracts, when co-incubated with the toxicant, protected significantly HepG2 cells against cell death. The methanolic extract, with a higher content of phenolic compounds than the water extract, conferred better protection in this in vitro model of oxidative stress with liver cells.”  A 2006 study Phenolic compounds protect HepG2 cells from oxidative damage: relevance of glutathione levels compared the antioxidant capabilities of several polyphenols  “If the effects of quercetin are given the reference value 1, the compounds rank in the following order according to inhibition of cell death: luteolin (4.0) > quercetin (1.0) > rosmarinic acid (0.34) > luteolin-7-glucoside (0.30) > caffeic acid (0.21). The results underscore the importance of the compound’s lipophilicity in addition to its antioxidant potential for its biological activity.”

Rosemary and oregano my offer several health benefits associated with rosmarinic acid content.  The 2006 publication Antioxidant and antimicrobial activities of rosemary extracts linked to their polyphenol composition concludes “Carnosic acid and rosmarinic acid may be the main bioactive antimicrobial compounds present in rosemary extracts. From a practical point of view, rosemary extract may be a good candidate for functional foods as well as for pharmaceutical plant-based products.”   The 1999 publication Pharmacology of rosemary (Rosmarinus officinalis Linn.) and its therapeutic potentials “Rosmarinic acid is well absorbed from gastrointestinal tract and from the skin. It increases the production of prostaglandin E2 and reduces the production of leukotriene B4 in human polymorphonuclear leucocytes, and inhibits the complement system. It is concluded that rosemary and its constituents especially caffeic acid derivatives such as rosmarinic acid have a therapeutic potential in treatment or prevention of bronchial asthma, spasmogenic disorders, peptic ulcer, inflammatory diseases, hepatotoxicity, atherosclerosis, ischaemic heart disease, cataract, cancer and poor sperm motility.”

A 2007study The effects of essential oils and aqueous tea infusions of oregano (Origanum vulgare L. spp. hirtum), thyme (Thymus vulgaris L.) and wild thyme (Thymus serpyllum L.) on the copper-induced oxidation of human low-density lipoproteins looked at “the antioxidative capacity effect of essential oils and aqueous tea infusions obtained from oregano, thyme and wild thyme on the oxidation susceptibility of low-density lipoproteins (LDL).  — The strong protective effect of aqueous tea infusions is proposed to be the consequence of large amounts of polyphenols, namely rosmarinic acid and flavonoids (quercetin, eriocitrin, luteolin-7-O-glucoside, apigenin-7-O-glucoside, luteolin, apigenin), with the most pronounced effect in the case of oregano.”  This is interesting and would seem to be a good argument for drinking oregano tea(ref), something I have never quite done.

Both water-soluble oil-soluble extracts of rosemary are commercially available.  The 2009 publication In vitro antimicrobial and antioxidant activity of commercial rosemary extract formulations concludes “Reducing power and free radical scavenging effectiveness was higher in water-soluble formulations, according to their higher total phenolic content, but in an aqueous emulsion system of linoleic acid, they exhibited lower antioxidant activity. This correlated well with the higher efficiency of antimicrobial activity of oil-soluble formulations, despite the lower total phenolic content of these extracts.”

The list of research showing potential benefits of rosmarinic acid and the herbs containing it seems to grow proportionally to the effort I have been putting into searching the literature on it.  For example Rosmarinic acid, a photo-protective agent against UV and other ionizing radiations, Radioprotective-antimutagenic effects of rosemary phenolics against chromosomal damage induced in human lymphocytes by gamma-rays  Antiviral and Anti-Inflammatory Effects of Rosmarinic Acid in an Experimental Murine Model of Japanese Encephalitis, Perilla-derived Rosmarinic Acid’s Effectiveness Against Hay Fever Confirmed, and Evaluation of clonal herbs of Lamiaceae species for management of diabetes and hypertension

I have written about several other phytochemicals in this blog and in my treatise ANTI-AGING FIREWALLS THE SCIENCE AND TECHNOLOGY OF LONGEVITY.  See, for example, yesterday’s post Nrf2 and cancer chemoprevention by phytochemicals. Who would think that, after a bout of dental x-rays, eating a slice of pizza with a generous layer of oregano sprinkled on it could be a good thing to do from a radioprotective viewpoint?

A cluster of research reports has appeared during the last few years looking at  mechanisms through which substances rich in phytochemicals (e.g. coffee, chocolate, turmeric, olive oil, broccoli, red hot peppers, green tea, garlic, blueberries, rosemary, oregano, sage) are cancer-preventative. While these foods have been studied for many years a new focal point has been moving to center stage – study of what these substances are doing in terms of gene expression as a key to understanding their therapeutic value.  The 2005 paper Dietary cancer-chemopreventive compounds: from signaling and gene expression to pharmacological effects articulates this emerging viewpoint.  “The process of cancer development (carcinogenesis leading to advanced metastasized cancers) in humans generally takes many years through initiation, promotion and progression. Because advanced metastasized cancers are almost impossible to treat, cancer chemoprevention for the control and containment of early cancer development is highly desirable. Recent studies have provided strong evidence that many daily-consumed dietary compounds possess cancer-protective properties that might interrupt the carcinogenesis process. These properties include the induction of cellular defense detoxifying and antioxidant enzymes, which can protect against cellular damage caused by environmental carcinogens or endogenously generated reactive oxygen species. These compounds can also affect cell-death signaling pathways, which could prevent the proliferation of tumor cells.”

One master activator of antioxidant and anticancer genes appears to be Nuclear factor-erythroid-2-related factor 2 (Nrf2). The sequence of events involved in phytochemical chemoprevention mediated by Nrf2 is complex and is summarized in the 2008 publication Nrf2 as a master redox switch in turning on the cellular signaling involved in the induction of cytoprotective genes by some chemopreventive phytochemicals.  “A wide array of dietary phytochemicals have been reported to induce the expression of enzymes involved in both cellular antioxidant defenses and elimination/inactivation of electrophilic carcinogens. Induction of such cytoprotective enzymes by edible phytochemicals largely accounts for their cancer chemopreventive and chemoprotective activities.” For those of you who have a taste for molecular biology, that document goes on to explain “Nuclear factor-erythroid-2-related factor 2 (Nrf2) plays a crucial role in the coordinated induction of those genes encoding many stress-responsive and cytoptotective enzymes and related proteins. These include NAD(P)H:quinone oxidoreductase-1, heme oxygenase-1, glutamate cysteine ligase, glutathione S-transferase, glutathione peroxidase, thioredoxin, etc. In resting cells, Nrf2 is sequestered in the cytoplasm as an inactive complex with the repressor Kelch-like ECH-associated protein 1 (Keap1). The release of Nrf2 from its repressor is most likely to be achieved by alterations in the structure of Keap1. Keap1 contains several reactive cysteine residues that function as sensors of cellular redox changes. Oxidation or covalent modification of some of these critical cysteine thiols would stabilize Nrf2, thereby facilitating nuclear accumulation of Nrf2. After translocation into nucleus, Nrf2 forms a heterodimer with other transcription factors, such as small Maf, which in turn binds to the 5′-upstream CIS-acting regulatory sequence, termed antioxidant response elements (ARE) or electrophile response elements (EpRE), located in the promoter region of genes encoding various antioxidant and phase 2 detoxifying enzymes. Certain dietary chemopreventive agents target Keap1 by oxidizing or chemically modifying one or more of its specific cysteine thiols, thereby stabilizing Nrf2. In addition, phosphorylation of specific serine or threonine residues present in Nrf2 by upstream kinases may also facilitate the nuclear localization of Nrf2. Multiple mechanisms of Nrf2 activation by signals mediated by one or more of the upstream kinases, such as mitogen-activated protein kinases, phosphatidylionositol-3-kinase/Akt, protein kinase C, and casein kinase-2 have recently been proposed.”

Two signaling pathways frequently mentioned in this blog, the MAPK/ERK and PI3K/Akt pathways, appeared to be involved as pointed out in the 2009 publication Salvia Fruticosa, Salvia Officinalis, and Rosmarinic Acid Induce Apoptosis and Inhibit Proliferation of Human Colorectal Cell Lines: The Role in MAPK/ERK Pathway. “In the present study, the antiproliferative and proapoptotic effects of water extracts of Salvia fruticosa (SF) and Salvia officinalis (SO) and of their main phenolic compound rosmarinic acid (RA) were evaluated in two human colon carcinoma-derived cell lines, HCT15 and CO115, which have different mutations in the MAPK/ERK and PI3K/Akt signalling pathways. — Our results show that SF, SO, and RA induce apoptosis in both cell lines, whereas cell proliferation was inhibited by the two sage extracts only in HCT15. SO, SF, and RA inhibited ERK phosphorylation in HCT15 and had no effects on Akt phosphorylation in CO115 cells. The activity of sage extracts seems to be due, at least in part, to the inhibition of MAPK/ERK pathway.” 

Phytochemical cancer chemoprevention may involve a number of additional pathways besides Nrf2 .  A number of phytosubstances are powerful anti-inflammatories for example and this may play a role in their control of cancer.  Bromelain, ginger, curcumin, aswagandah and boswellia are in this category.  Inhibition of TNFalpha-activated NF-kappaB signaling may also play an important role in preventing cancer activation as pointed out in my treatise.  A number of herbal substances that are NF-kappaB inhibitors are in my combined anti-aging firewall. 

The use of herbal substances for cancer chemoprevention is receiving a lot of attention, especially now that the chains of molecular activities initiated by these traditional substances are starting to be understood.  The 2007 publication Chemopreventive herbal anti-oxidants: current status and future perspectives states “Cancer chemoprevention is fast becoming a lucrative approach for controlling cancer. Carcinogenesis being a complex multi-step, multi-factorial process, a number of chemopreventive interventions can be employed. These strategies are generally directed against two broad events of carcinogenesis viz., initiation and promotion/progression. Anti-initiation interventions principally involve inhibition of carcinogen activation, scavenging of free radicals and reactive carcinogen metabolites along with enhanced detoxification of carcinogens by modulating cellular metabolism. Anti-promotion strategies involve attenuation of enhanced cellular proliferation along with induction of cellular apoptosis and differentiation. Dietary agents or herbal anti-oxidants due to low toxicity and relative safety are promising chemopreventive agents.” 

Other publications on cancer chemoprevention include Comprehensive review of cancer chemopreventive agents evaluated in experimental carcinogenesis models and clinical trials, Chemopreventive effects of natural dietary compounds on cancer development,  Organosulfur compounds in cancer chemoprevention, Cancer prevention by natural compounds, Cruciferous vegetables and cancer prevention, Cruciferous vegetables and human cancer risk: epidemiologic evidence and mechanistic basis, Cruciferous vegetables: cancer protective mechanisms of glucosinolate hydrolysis products and selenium. Following the hyperlinks to and from these publications will lead to many more.Those of you familiar with my treatise ANTI-AGING FIREWALLS – THE SCIENCE AND TECHNOLOGY OF LONGEVITY  know that I suggest consumption of phytochemical-rich foods like blueberries, walnuts, chocolate and green tea and suggests taking a substantial number of phytosubstance supplements including resveratrol, curcumin, boswellia, ashwagandah, pycnogenol, green tea extract, olive leaf extract, lycopene, allicin and  OPC grape seed extract.  The blog post Phytochemicals – focus on caffeic acid  looks at one important phytochemical in depth, the one that is in coffee. And the post Health and longevity benefits of dark chocolate looks at another of my favorite phytosubstances.

Another action of Nrf2 is protection of arteries from fluid sheer stress generated by blood flow.  The 2009 publication Regulation of shear-induced nuclear translocation of the Nrf2 transcription factor in endothelial cells describes how sheer stress induces nuclear translocation of Nrf2 which restores laminar flow.  The paper concludes “Our data suggest that the atheroprotective effect of laminar flow is partially attributed to Nrf2 activation which results in ARE-mediated gene transcriptions, such as HO-1 expression, that are beneficial to the cardiovascular system.”  Several other papers have been written on this effect  such as the 2007 paper Shear stress stabilizes NF-E2-related factor 2 and induces antioxidant genes in endothelial cells: role of reactive oxygen/nitrogen species.

For a personal story from my childhood involving two phytosubstances and cancer, see the blog post Red wine, hot peppers and my uncle Gigi.

For a great many years the medical establishment warned everyone of dire consequences that could result from taking large doses of vitamin D.  The daily maximum of 400iu was the strict acceptable limit to avoid vitamin D toxicity.  In recent years the consensus of the medical establishment has flipped as a result of many studies showing dire consequences of too little vitamin D and strong benefits of taking it as a supplement.  As I state in my treatise “A recent eight-year study of 3,258 men and women  indicates that the higher the blood level of Vitamin D, the less is the chance of dying from heart disease – and the less the chance of dying from a number of other diseases as well.”  — “Today there are over 80 population and laboratory studies indicating that vitamin D can reduce incidences of and mortality due to multiple kinds of cancer with reductions of 50% or more in some cases.  The biological impact of this substance is far from simple; it activates 200 or more human genes and has differential affects in regulating cancer cells with respect to cell proliferation, apoptosis and differentiation.  It also regulates angiogenesis.  Several studies of nursing home and residential care residents show that people taking vitamin D supplements suffer fewer falls – the reduction being between 23% to 53%.  And I have already mentioned Vitamin D’s role with respect to reducing heart disease fatalities.   Daily doses of 1,000 or 2,000iu are now thought to be harmless and often recommended for older people.“  Advising people to take even larger doses is not unusual now.

There is a possibility the viewpoint may be flipping again, at least for patients with certain genetic defects.  Mouse studies suggest that excess levels of vitamin D under certain situations may lead to premature aging.   The concern is current.  The 2009 publication Vitamin D and aging states “Recent studies using genetically modified mice, such as FGF23-/- and Klotho-/- mice that exhibit altered mineral homeostasis due to a high vitamin D activity showed features of premature aging that include retarded growth, osteoporosis, atherosclerosis, ectopic calcification, immunological deficiency, skin and general organ atrophy, hypogonadism and short lifespan. The phenotype reversed by normalizing vitamin D and/or mineral homeostasis.” 

The story is also summarized in the abstract of the 2006 publication Hypervitaminosis D and premature aging: lessons learned from Fgf23 and Klotho mutant mice. “The essential role of low levels of vitamin D during aging is well documented. However, possible effects of high levels of vitamin D on the aging process are not yet clear. Recent in vivo genetic-manipulation studies have shown increased serum level of vitamin D and altered mineral-ion homeostasis in mice that lack either fibroblast growth factor 23 (Fgf23) or Klotho (Kl) genes. These mice develop identical phenotypes consistent with premature aging. Elimination or reduction of vitamin-D activity from Fgf23 and Kl mutant mice, either by dietary restriction or genetic manipulation could rescue premature aging-like features and ectopic calcifications, resulting in prolonged survival of both mutants. Such in vivo experimental studies indicated that excessive vitamin-D activity and altered mineral-ion homeostasis could accelerate the aging process.”  

So, these studies have led me to inquire about what is involved and what is the danger of taking 2000 to 4000iu of vitamin D3 a day as I have personally been doing.  My bottom line is that you should be particularly concerned if you are a Klotho-knockout mouse, a FGF23 knockout mouse or a vitamin D receptor knockout mouse.  If you happen to be in one of the first two categories,  you may be able to live longer by immediately ceasing to take vitamin D supplements.  Also, you could have a Hypervitaminosis D problem if your natural FGF23 or Klotho expression is sub-par.

My remarks are based on the fact that the vitamin D research is based on experiments with Klotho knockout and FGF23 knockout (FGF23-/- and Klotho-/-) mice.   Conceivably, similar D hypervitaminosis could happen in people with Klotho or FGF23 deficiencies.  See the recent post Klotho anti-aging gene in the news.  I have reviewed the following papers which describe mouse studies on laboratory animals with the characteristics in parentheses:

The 1997 paper Mutation of the mouse Klotho gene leads to a syndrome resembling ageing (mutated Klotho gene)

The 2002 publication Association of human aging with a functional variant of Klotho (mutated Klotho gene)

The 2004 paper Klotho is a serum factor related to human aging (FGF23 null mice)

The 2006 publication Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process (FGF23 null mice)

The 2006 paper Genetic ablation of vitamin D activation pathway reverses biochemical and skeletal anomalies in Fgf-23-null animals (FGF23 null mice)

The 2006 publication Hypervitaminosis D and premature aging: lessons learned from Fgf23 and Klotho mutant mice (FGF23 null or Klotho null mice)

The 2007 paper Premature aging in Klotho mutant mice: cause or consequence? (FGF23 null or Klotho null mice)

The 2008 paper FGF-23-Klotho signaling stimulates proliferation and prevents vitamin D-induced apoptosis (FGF23 null or Klotho null mice)

The 2009 publication Vitamin D and aging (FGF23 null or Klotho null mice) 

All these studies related to Hypervitaminosis D were based on working with mice in which either Klotho or FGF23 or both were knocked out.  It seems that when the transduction pathways initiated by FGF23 and Klotho are working well, apoptosis caused by excessive systemic vitamin D and resulting tissue atrophy is avoided.    

Another  2009 study is of particular interest because the mice were ones with the vitamin D receptor knocked out instead of Klotho or FGF23. The publication Premature aging in vitamin D receptor mutant mice  (vitamin D receptor (VDR) knockout mice)  states “Overall, VDR KO mice showed several aging related phenotypes, including poorer survival, early alopecia, thickened skin, enlarged sebaceous glands and development of epidermal cysts.”  “Since the phenotype of aged VDR knockout mice is similar to mouse models with hypervitaminosis D(3), our study suggests that VDR genetic ablation promotes premature aging in mice, and that vitamin D(3) homeostasis regulates physiological aging.”   

Knocking out VDR seems capable of producing the same pro-aging affect as hypervitaminosis D(3).  I believe the conclusion that “vitamin D(3) homeostasis regulates physiological aging” could be of profound importance if it is born out. 

Since I have normal Klotho and FGF23 expression as far as I know, I am not personally worried about Hypervitaminosis D at the moment.

Since its discovery in 1997 the Klotho gene has been known to be involved with longevity.  I came across a recent news article describing research linking expression of the gene to reduction in hypertension, and this led me to look into what is known about the gene.  As reported here, Klotho appears to have a lot of interesting properties.

“The Klotho gene codes for a transmembrane protein that, in addition to other effects, provides some control over the sensitivity of the organism to insulin and appears to be involved in aging. Its discovery was documented in 1997 by Kuro-o et al.^[1] The name of the gene comes from Klotho or Clotho, one of the Moirae, or Fates, in Greek mythology(ref).”  In mice at least the Klotho protein acts like a hormone.  It circulates through the blood and binds to cells.

The newly-reported publication is Klotho Gene Delivery Prevents the Progression of Spontaneous Hypertension and Renal Damage.  “Researchers, led by principal investigator Zhongjie Sun, tested the effect of an anti-aging gene called Klotho on reducing hypertension. They found that by increasing the expression of the gene in laboratory models, they not only stopped blood pressure from continuing to rise, but succeeded in lowering it. Perhaps most impressive was the complete reversal of kidney damage, which is associated with prolonged high blood pressure and often leads to kidney failure(ref).”  The experiment was on laboratory rats where the gene was delivered by a virus vector.  “This is the first study showing that a decline in Klotho protein level may be involved in the progression of hypertension and kidney damage, Sun said. With age, the Klotho level decreases while the prevalence of hypertension increases. —  Researchers used one injection of the Klotho gene in hypertensive research models and were able to markedly reduce blood pressure by the second week. It continued to decline steadily for the length of the project – 12 weeks. The Klotho gene was delivered with a safe viral vector that is currently used for gene therapy. The virus is already approved by the U.S. Food and Drug Administration for use in humans(ref).”  Of course, it is hoped that the research will at some point lead to a Klotho therapy for humans for hypertension and associated kidney damage.

Studies relating Klotho to aging started to appear soon after its discovery.  Most of these are based on working with rats or mice.  Some of the earlier publications related deficiency of Klotho to classical indicators of aging.  The 1977 publication Mutation of the mouse Klotho gene leads to a syndrome resembling ageing states “A defect in Klotho gene expression in the mouse results in a syndrome that resembles human ageing, including a short lifespan, infertility, arteriosclerosis, skin atrophy, osteoporosis and emphysema. —  The Klotho gene product may function as part of a signaling pathway that regulates ageing in vivo and morbidity in age-related diseases.” 

The 2002 publication Association of human aging with a functional variant of Klotho concludes “These results suggest that the KL-VS allele influences the trafficking and catalytic activity of Klotho, and that variation in Klotho function contributes to heterogeneity in the onset and severity of human age-related phenotypes.”  The study suggested that one of the aging mechanisms that may be accelerated by insufficient expression of Klotho is the buildup of advanced glycation endproducts (AGEs). “Multiple models of aging invoke accelerated or excessive posttranslational modification of proteins including glycation. Resultant advanced glycation end-products (AGEs) elicit a wide range of responses that have been proposed to contribute to many age related phenotypes, including atherosclerosis, Alzheimer’s disease, diabetic complications, and microvascular changes (29). It is possible that the proposed glycosidase activity of Klotho retards the accumulation of AGEs.” Note that Tissue Glycation is an important theory of aging discussed in my treatise.

Research articles implicate low levels of Klotho expression with endothelial dysfunction, pulmonary emphysema,  impairment of osteoblast and osteoclast differentiation, cognition impairment and other disease processes in mouse models.

The 2004 paper Klotho is a serum factor related to human aging looks at Klotho protein in human serum in 112 individuals.  “ The population aged from 0 to 91 years screened by ELISA revealed that the level of serum KL declined while age increased, though each individual level was variable and that the trend of decreasing in serum KL had no difference in sex. – Conclusion: Our data suggest that KL is a serum factor related to human aging.”

Klotho’s actions and the channels it works through are complex.  From the 2006 paper Toward a better understanding of Klotho: “Suggested functions of Klotho are (i) a fundamental regulator of calcium homeostasis, namely, a cofactor for the fibroblast growth factor (FGF) receptor 1c in FGF23 signaling and a regulator of parathyroid hormone secretion; (ii) a hormone that interferes with the intracellular signaling of insulin and insulin-like growth factor-1 (IGF-1); and (iii) a beta-glucuronidase that activates the transient receptor potential ion channel TRPV5 by trimming its sugar moiety.”   

As to how Klotho may impact on longevity: a) I have already mentioned its actions in averting tissue glycation, b) the IGF-1 pathway has long been known to be associated with longevity and is that affected by calorie restriction, and c) Klotho promotes the body’s antioxidant defenses.

Klotho expression is also important for averting premature aging due to overexpression of Vitamin D.  The mechanism is associated with its function in regulating FGF23.  (YES my reader friends, in animal model experiments, overexpression of vitamin D leads to premature aging.  I will cover that issue in a separate blog post).  The 2002 publication Klotho, a gene related to a syndrome resembling human premature aging, functions in a negative regulatory circuit of vitamin D endocrine system states “These observations suggest that Klotho may participate in a negative regulatory circuit of the vitamin D endocrine system, through the regulation of 1alpha-hydroxylase gene expression.” The 2008 paper FGF-23-Klotho signaling stimulates proliferation and prevents vitamin D-induced apoptosis states “We show that the signal transduction pathways initiated by FGF-23-Klotho prevent tissue atrophy by stimulating proliferation and preventing apoptosis caused by excessive systemic vitamin D. Because serum levels of active vitamin D are greatly increased upon genetic ablation of Fgf-23 or Klotho, we find that these molecules have a dual role in suppression of apoptotic actions of vitamin D through both negative regulation of 1alpha-hydroxylase expression and phosphoinositide-3 kinase-dependent inhibition of caspase activity.”

A number of other papers also deal with the involvement of Klotho and FGF23 in vitamin D mediated premature aging, for example Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process concludes “our data support a new model of interactions among Fgf-23, vitamin D, and Klotho, a gene described as being associated with premature aging process.”  

Besides Klotho defects being implicated in disease processes there is some evidence that overexpression of Klotho could be life-extending.  For example, the 2005 paper Suppression of Aging in Mice by the Hormone Klotho states “Here, we show that overexpression of Klotho in mice extends life span. Klotho protein functions as a circulating hormone that binds to a cell-surface receptor and represses intracellular signals of insulin and insulin-like growth factor 1 (IGF1), an evolutionarily conserved mechanism for extending life span. Alleviation of aging-like phenotypes in Klotho-deficient mice was observed by perturbing insulin and IGF1 signaling, suggesting that Klotho-mediated inhibition of insulin and IGF1 signaling contributes to its anti-aging properties. Klotho protein may function as an anti-aging hormone in mammals.” The point is reinforced in several other publications.

The link of Klotho to insulin is further reinforced by a 2007 study that involved comparing the expression of genes in young and old brains(ref).  “They observed that the levels of Klotho in the brain showed a striking decrease with aging. The association between Klotho and aging prompted Abraham’s group to investigate the regulation of Klotho further. These studies lead to the observation that secretion of Klotho is regulated by insulin. — To their surprise, they found that insulin, a hormone usually associated with diabetes, increases significantly the levels of secreted Klotho. The reason this finding is important is because excess insulin has been previously implicated in a biochemical pathway that is associated with a decreased life span and elevated oxidative stress. — In addition, this observation provides a potentially pivotal link between Klotho and sugar metabolism, and raises an intriguing relationship between Klotho and type II diabetes, commonly known as late onset diabetes. The authors are proposing a novel mechanism of action for Klotho whereby insulin increases Klotho secretion, i.e., activity, and in turn, the secreted Klotho inhibits insulin’s actions in the cell, which are known to be detrimental when insulin is in excess(ref).”

One viewpoint is that Klotho derives much of its anti-aging capability from the protein acting “by increasing the cell’s ability to detoxify harmful reactive oxygen species (ref).  “Using cultured cells and transgenic mice, the researchers showed that Klotho increases resistance to oxidative stress.   “Increased longevity is always associated with increased resistance to oxidative stress,” explains Kuro-o (the man who discovered Klotho). “Oxidative stress causes the accumulation of oxidative damage to important biological macromolecules such as DNA, lipids, and proteins that would result in functional deterioration of the cell, which eventually causes aging(ref).”  Of course, this viewpoint is consistent with the classical Oxidative Damage theory of aging.

A 2007 report is entitled Obesity May Be Associated With A Relative Of Anti-aging Gene, Klotho.  Differences in how to interpret results with Klotho knockout mice has also engendered controversy about its anti-aging capacities such as described in a blog article I found entitled Controversial Klotho in cancer.

Despite that it has been studied for over a dozen years, the exact anti-aging mechanisms of Klotho still are not clearly identified.  The 2008 paper Klotho as a regulator of oxidative stress and senescence states “The Klotho gene functions as an aging-suppressor gene that extends life span when overexpressed and accelerates aging-like phenotypes when disrupted in mice. — The secreted Klotho protein can regulate multiple growth factor signaling pathways, including insulin/IGF-1 and Wnt, and the activity of multiple ion channels. Klotho protein also protects cells and tissues from oxidative stress, yet the precise mechanism underlying these activities remains to be determined. Thus, understanding of Klotho protein function is expected to provide new insights into the molecular basis for aging, phosphate/vitamin D metabolism, cancer and stem cell biology.”                          

As time goes on we will doubtlessly be hearing more about Klotho.

An interesting report came to my attention relating to telomeres in pythons, and this set me off for the umpteenth time pursuing further research and thoughts about cell senescence, telomere lengths and telomerase. I share that all here.

First of all, about water pythons (Liasis fuscus), snakes that grow to about 2 meters in length.  They are found in flood plains in Australia and New Guinea and are relatively long-lived.  The just-released study report Short Telomeres in Hatchling Snakes: Erythrocyte Telomere Dynamics and Longevity in Tropical Pythons starts out with  “In the present study we explore whether age- and sex-specific telomere dynamics affect life span in a long-lived snake, the water python (Liasis fuscus).”  “– Erythrocyte TL (telomere length) was measured using the Telo TAGGG TL Assay Kit (Roche). In contrast to other vertebrates, TL of hatchling pythons was significantly shorter than that of older snakes. However, during their first year of life hatchling TL increased substantially. While TL of older snakes decreased with age, we did not observe any correlation between TL and age in cross-sectional sampling. In older snakes, female TL was longer than that of males. When using recapture as a proxy for survival, our results do not support that longer telomeres resulted in an increased water python survival/longevity.” – “In fish high telomerase activity has been observed in somatic cells exhibiting high proliferation rates. Hatchling pythons show similar high somatic cell proliferation rates. Thus, the increase in TL of this group may have been caused by increased telomerase activity. In older humans female TL is longer than that of males. This has been suggested to be caused by high estrogen levels that stimulate increased telomerase activity. Thus, high estrogen levels may also have caused the longer telomeres in female pythons. The lack of correlation between TL and age among old snakes and the fact that longer telomeres did not appear to affect python survival do not support that erythrocyte telomere dynamics has a major impact on water python longevity.” 

OK.  Taken at face value, the study says to forget about the Telomere shortening theory of aging at least as far as these snakes are concerned.  What set me off thinking in the report, however, was the statement “However, other studies have demonstrated that TL does not affect survival among old humans. Furthermore, replicative senescence has been shown to be induced by changes in the protected status of the telomeres rather than the loss of TL.”  This is the opposite of the party line believed by most people doing telomerase activation as an anti-aging measure.  So I decided to pursue the author’s citations to check out these assertions. 

The 2005 study Telomere length in white blood cells is not associated with morbidity or mortality in the oldest old: a population-based study which studied 598 participants concludes “Telomere length at baseline was not predictive for mortality (P > 0.40 for all-cause, cardiovascular causes, cancer or infectious diseases, Cox regression for gender-adjusted tertiles of telomere length) or for the incidence of dementia (P = 0.78). Longitudinally, telomere length was highly unstable in a large fraction of participants. We conclude that blood monocyte telomere length is not a predictive indicator for age-related morbidity and mortality at ages over 85 years, possibly because of a high degree of telomere length instability in this group.” 

Another 2006 study No association between telomere length and survival among the elderly and oldest old looks at a different population and confirms the result. “Telomere length was measured as mean terminal restriction fragment length on blood cells from 812 persons, age 73 to 101 years, who participated in population-based surveys in 1997-1998. Among the participants were 652 twins. The participants were followed up through the Danish Civil Registration system until January 2005, at which time 412 (51%) were dead. RESULTS: Univariate Cox regression analyses revealed that longer telomeres were associated with better survival (hazard ratios = 0.89 [95% confidence interval = 0.76-1.04] per 1 kb in males and 0.79 [0.72-0.88] per 1 kb in females, respectively). However, including age in the analyses changed the estimates to 0.97 (0.83-1.14) and 0.93 (0.85-1.03), respectively.”  — “CONCLUSION: This longitudinal study of the elderly and oldest old does not support the hypothesis that telomere length is a predictor for remaining lifespan once age is controlled for.”  

A number of other recent studies also question the relationship of telomere lengths to mortality.  “The question remains as to whether telomere dynamics is a determinant or merely a predictor of human biological age over and above chronological ageing. Although several reports have suggested a link between telomere attrition and ageing phenotypes and disorders, both reference values and a complete set of determinants are missing(ref).”  What is happening to the Telomere shortening theory of aging?  In all fairness there are a number of other studies that relate telomere shortening to a number of disease processes. 

Another report cited in the python paper is the 2002 study Senescence Induced by Altered Telomere State, Not Telomere Loss  which seems to be another attack on those pursuing longevity through telomerase activation.  “Here, we report that overexpression of TRF2, a telomeric DNA binding protein, increased the rate of telomere shortening in primary cells without accelerating senescence. TRF2 reduced the senescence setpoint, defined as telomere length at senescence, from 7 to 4 kilobases. TRF2 protected critically short telomeres from fusion and repressed chromosome-end fusions in presenescent cultures, which explains the ability of TRF2 to delay senescence. Thus, replicative senescence is induced by a change in the protected status of shortened telomeres rather than by a complete loss of telomeric DNA.”  Google shows this study is cited by 403 others. 

Many of these studies deal with telomerase binding proteins and the very complex processes involved in telomere elongation.  While there seems consensus on the importance of telomere states I am not at all clear that the simplistic conclusion of the title of this report is widely endorsed.  Many publications express statements such as “loss of telomere integrity is a major trigger for the onset of premature senescence under mild chronic oxidative stress(ref).”  Note that in my treatise I have expanded the 12^th theory of aging to go beyond simple telomere shortening to read Telomere Shortening and Damage.

My own thoughts

1.      Cell senescence is a bad thing and is postponed or avoided under healthy conditions even in cells that have replicated many times by cell signaling that either a) naturally activates telomerase at the last minute, or b) activates telomerase binding factors to delay senescence such as suggested above, or c) triggers apoptosis.

2.     Loss of cells that have replicated many times due to healthy apoptosis need not affect health as long as there is a ready and able contingent of progenitor and stem cells to replace them (re the stem cell supply chain).

3.     Telomerase activation by using exogenous telomerase activators may or may not work to extend telomeres of somatic cells, depending on telomere binding factor complexities.  It may or may not work to thwart cell senescence.  The complex natural mechanisms that control telomere lengths may work to subvert telomere extension for many classes of cells.  Have you noticed that now, in year 2 of people striving to lengthen their telomeres via TA-2, astragaloside, etc. the almost complete absence of published reports of people that have demonstrably lengthened their telomeres? 

4.     Even if the average lengths of somatic cell telomeres can be increased by telomerase activation, this by itself may or may not have an effect on human longevity. (This statement could get me burned at the virtual stake in certain longevity circles, except for the following statement.)

5.     Telomerase activation may nonetheless have a strong positive effect in supporting healthy operation of the stem cell supply chain and may therefore be worth doing despite its effects on telomere lengths. See the blog post Revisiting telomere shortening yet-again.

For reference purposes, a list of my previous writings related to telomerase can be found in this post .

I have written about cancer stem cells several times in this blog, but many oncologists and cancer researchers still see cancer stem cells mainly as hypothetical entities whose relevance if not very-existence is questionable.  A recent article in Gen points out that a number of pharmaceutical companies are betting big on cancer therapies based on going after cancer stem cells.

As I wrote in my July 2009 post On cancer stem cells, most cancer therapies are based on killing cancer cells – as many cells as possible.  But cancers frequently and persistently recur after bouts of radiation or chemotherapy.  The culprit is thought to be cancer stem cells, where any surviving ones simply go about making new cancer cells.  A new therapeutic concept is therefore to focus on killing the cancer stem cells.  “While normal stem cells are essential for development, play a key role in tissue maintenance, and aid in repair, cancer stem cells are believed responsible for tumorigenesis, metastases, and cancer recurrence(ref).”  I reported further research regarding cancer stem cells in my August 2009 blog post Update on cancer stem cells.

It turns out the way to do this is to target the same signaling pathways involved in the proliferation and differentiation of normal stem cells, pathways I have discussed previously in this blog. 

Notch is one such pathway which I discussed in the post On cancer stem cells.  As reported in Gen: “Different points in the (Notch) pathway have been targeted for drug development. OncoMed Pharmaceuticals’ OMP-21M18 is an antibody that blocks signals by binding to Delta-like ligand. The drug, which is in a clinical trial involving patients with advanced solid tumors, is part of a $1.4 billion collaboration with GlaxoSmithKline.  Merck and Roche have inhibitors to γ-secretase that cleaves the Notch receptor releasing the Notch intracellular domain, a transcription factor. Both companies’ drugs are in early testing against solid tumors. Finally, Trojantec is targeting the Notch pathway with a truncated version of Mastermind, a coactivator involved in chromatin-specific transcription. The drug may prove useful against tumors that overexpress Notch signaling components(ref).”  The role of Notch signaling in stem cell proliferation and differentiation was touched on in my blog post Niche, Notch and nudge.

PI3K/Akt is another pathway being targeted.  “The PI3K/Akt pathway’s importance in cancer is partly attributable to PI3K’s (phosphatidylinositol 3-kinase’s) association with oncogenic growth factor receptors, notably for epidermal growth factor, platelet-derived growth factor, and mesenchymal transition factor. The pathway is also prone to mutations associated with oncogenesis, including changes in the catalytic subunit of PI3K that occur in prostate, breast, endometrium, urinary tract, and colon cancers. — Similarly, mutations of the lipid phosphatase PTEN that normally serves to deactivate the PI3K/Akt pathway are found in cancers of the endometrium, brain, skin, and prostate, while mutations in the protein kinase Akt, which is downstream of PI3K, are overexpressed in head and neck squamous cell carcinoma, and in pancreatic and ovarian cancers. Eight drugs targeting the PI3K/Akt pathway are in clinical trials(ref).”  I have mentioned the P13K/Akt/mTOR and its relationship to stem cells in several posts including More mTOR links to aging theories.

The Hedgehog signaling pathway is another one being targeted by new drugs in the pipeline.   “The Hedgehog pathway provides an intercellular regulatory mechanism that serves essential functions in the normal proliferation and differentiation of stem cells. Mutations in this pathway figure in basal cell carcinoma, medulloblastoma, and other malignancies. Three drugs that interfere with hedgehog signaling are in clinical trials—two, Infinity Pharmaceuticals’ IPI-926 and Genentech/Curis’ GDC-0449, are derivatives of cyclopamine, which has been studied extensively(ref).”

Heavy players in the pharma industry are betting big on new therapies for going after cancer stem cells.  Perhaps more cancer researchers should start watching where the “smart money” is flowing.

This post reviews some key research findings regarding liver regeneration and discusses what is known about the mechanisms involved.   It turns out that a bunch of my favorite blog topics are involved: telomerase, stem/progenitor cells, mTOR signaling, MAPK signaling, NOTCH signaling and NF-kappaB activation. 

“The liver is the largest and most metabolically complex organ in humans(ref),”  it has a complex internal structure(ref), and it performs many diverse tasks required for the health of other body systems(ref).  Further, the human liver has a remarkable capacity for self-regeneration when damaged or when even up to 80% of it is removed by surgery(ref).  Unlike almost any other organ in humans, it can simply grow back.  If a section of the liver is removed, as part of regeneration “normally quiescent hepatocytes undergo one or two rounds of replication to restore the liver mass by a process of compensatory hyperplasia(ref).”   Hepatocytes are the central cells involved in normal liver functioning making up 70% to 80% of the mass of the liver.  Compensatory hyperplasia simply means extra fast proliferation of cells to replace a lost part of the liver.  The amazing thing is how fast this happens.  In a study of regeneration in pig livers, maximum regenerative response occurred three days after up to 70% partial hepatectomy(ref).  “The timing and events of the regenerative response in the pig compared favorably with other animal models and the maximum regenerative response occurred on the third postoperative day, irrespective of the size of the partial hepatectomy.” 

Liver regeneration is not always possible depending on the nature and extent of damage, and the age of the patient involved.   According to the American Liver Foundation, around 17,000 Americans are currently on a waiting list for a liver transplant.  Understanding of what is involved in liver regeneration might assist in facilitating regeneration beyond that normally possible and possibly in developing regeneration strategies for other human organs.

·        Telomerase activation is centrally involved in liver regeneration

Since telomere shortening has long been known to limit organ regeneration(ref), it can be concluded a-priori that liver regeneration must involve the expression of telomerase.  The publication Regeneration in pig livers by compensatory hyperplasia induces high levels of telomerase activity tells an important part of the story.  In simple language, liver regeneration is accompanied by a burst of telomerase activation.  “Quiescent human hepatocytes exhibit very low or undetectable levels of telomerase activity. However, hepatocytes display a remarkable proliferative capability following liver injury. To investigate whether liver regeneration by compensatory hyperplasia is associated with telomerase activation, we measured telomerase activity in pig livers after 70 to 80% partial hepatectomy using a fully quantitative real-time telomeric repeat amplification protocol. In contrast to commonly studied inbred laboratory mouse strains, telomere length and telomerase activity in porcine tissues are comparable to humans. RESULTS: Following partial hepatectomy, histology revealed mitotic hepatocytes as marker for compensatory hyperplasia. As expected, there was no induction of inflammation. Telomerase activity increased significantly showing the highest levels (5-fold upregulation) in pigs treated with partial hepatectomy and hepatic decompression. Moreover, telomerase activity significantly correlated to the number of mitotic hepatocytes. CONCLUSION: Our data demonstrate telomerase activation in liver regeneration by compensatory hyperplasia in a large animal model with telomere biology comparable to humans. Telomerase activation may constitute a mechanism to protect proliferating liver cells against telomere shortening and oxidative stress.”  [Some of the research reported here is based on working with pig livers, good models of human livers. “Moreover, pig telomeres are comparable to those of humans regarding length and shortening during aging (ref)(ref). Because of these similarities, pigs have been utilized as model system to investigate telomerase regulation and telomere dynamics in mammalians(ref)(ref).”]

·        Telomerase activation possibly allows extensive mitosis (cell division) of hepatocytes

The publication Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential provides data with respect to fetal hepatocytes that may or may not apply to the mature working hepatocytes that remain in the working part of a partially destroyed liver.  The study was of fetal hepatocytes in-vitro.  “Telomerase-reconstituted cells were capable of preserving elongated telomeres, propagated in culture beyond replicative senescence for more than 300 cell doublings (to date), and maintained their liver-specific nature, as analyzed by a panel of hepatic growth factors, growth factor receptors, and transcription factors as well as albumin, glucose-6-phosphatase, glycogen synthesis, cytochrome P450 (CYP) expression profiles, and urea production. Moreover, the immortalized cells exhibited no oncogenicity, and no up-regulation of c-Myc was detected. The cells engrafted and survived in the liver of immunodeficient mice with hepatocellular gene expression.”  The extent to which this finding applies to mature hepatocytes in-vivo subject to niche signaling is unknown.

·        The differentiation of hepatic progenitor cells is involved.

An important question is “where do the new hepatocytes come from?  There are two possibilities: mitosis (cell division) of the hepatocytes already in the liver, and differentiation of hepatic progenitor cells to make new hepatocytes.  “Hepatic progenitor cells are immature epithelial cells that reside in the smallest ramifications of the biliary tree in human liver. These cells are capable of differentiating toward the biliary and the hepatocytic lineages. — an increased number of progenitor cells (referred to as “activation”) and differentiation of the same toward hepatocytes or bile duct epithelial cells, or both, is a component of virtually all human liver diseases. The extent of progenitor cell activation and the direction of differentiation are correlated with the severity of the disease and the type of mature epithelial cell (hepatocyte or bile duct epithelial cell), respectively, that is damaged(ref).”

These findings are interesting but raised my thirst for answers to several other questions:  1.  What cells are making the telomerase (e.g. hepatic progenitor cells or hepatocytes)? 2.  What is the role of the telomerase in liver regeneration? 3.  To what extent is the liver regeneration due to mitosis of existing hepatocytes and to what extent is it due to differentiation of hepatic progenitor cells? 4.  What other stem cells might be involved, such as for renewing the supply of hepatic progenitor cells?  5. What kind of signaling is articulating the whole regeneration process?  6. What hope does all this offer for longevity?  So, I went on a literature quest for a couple of days.  I found a number of research papers that cast slivers and rays of light on some of the questions.

·        Normally by themselves, hepatocytes do not express telomerase

In fact the publication In vitro expansion of human hepatocytes is restricted by telomere-dependent replicative aging says  “As expected, untransduced PHH (proliferating human hepatocytes ) progressively lost telomeric repeats and arrested after 30-35 cell divisions with telomeres of less than 5 kilo bases. In comparison, telomerase-reconstituted PHH maintained elongated telomeres and continued to proliferate as shown by colorimetric assays and cell counts. In this study, telomere stabilization extended the proliferative capacity of in vitro proliferating human neonatal hepatocytes. Therefore, telomere attrition needs to be addressed when developing techniques to expand human hepatocytes.”

·        Liver regeneration capability declines with the age of the animal or person

An explanation is given in a publication entitled Aging Reduces Proliferative Capacities of Liver by Switching Pathways of C/EBPα Growth Arrest.  “The liver is capable of completely regenerating itself in response to injury and after partial hepatectomy. In liver of old animals, the proliferative response is dramatically reduced, the mechanism for which is unknown. The liver specific protein, C/EBPα, normally arrests proliferation of hepatocytes through inhibiting cyclin dependent kinases (cdks). We present evidence that aging switches the liver-specific pathway of C/EBPα growth arrest to repression of E2F transcription. We identified an age-specific C/EBPα-Rb-E2F4 complex that binds to E2F-dependent promoters and represses these genes. The C/EBPα-Rb-E2F4 complex occupies the c-myc promoter and blocks induction of c-myc in livers of old animals after partial hepatectomy. Our results show that the age-dependent switch from cdk inhibition to repression of E2F transcription causes a loss of proliferative response in the liver because of an inability to induce E2F target genes after partial hepatectomy providing a possible mechanism for the age-dependent loss of liver regenerative capacity.” No surprise since many studies show that c-myc plays critical roles in cell proliferation and differentiation.  

·        Liver regeneration involves complex signaling

This signaling has been studied for some time.  I have already mentioned the roles of E2F transcription and c-myc.  A 1998 paper Signal transduction during liver regeneration states “Following partial hepatectomy (PH), there is a rapid and highly orchestrated series of biochemical events which occur prior to cellular proliferation. Some of these events are presumably intimately linked with the eventual regeneration of the liver, whereas others are likely to be stress related or required for the continued differentiated function of the liver while regeneration is occurring.  The regulation of the AP-1 transcription factor c-Jun during hepatic regeneration has been studied here. — It is concluded that the stimulation of one-third or two-thirds PH (partial hepatectomy) activates JNK through a mechanism that requires TNFalpha, which phosphorylates the c-Jun activation domain in hepatocytes, resulting in enhanced transcription of AP-1-dependent genes. — the induction of NFkappaB during liver regeneration following PH appears to be a required event to prevent apoptosis and to allow for normal cell cycle progression.”  I comment that the induction of NF-kappaB is not surprising since such translocation of NF-kappaB into the cell nucleus is required to activate the expression of a number of growth-related genes.

·        MAPK, mTOR  and Notch pathways are involved in liver regeneration, suggesting that stem/progenitor cell differentiation plays an important role.

The publication PI3K-FRAP/mTOR pathway is critical for hepatocyte proliferation whereas MEK/ERK supports both proliferation and survival suggests intimate involvement in liver regeneration of two pathways that have been discussed in this blog: mTOR and MAPK/ERK, and lends further weight to the argument that differentiation of stem/progenitor cells may be critically involved.  The blog post More mTOR links to aging theories links mTOR expression to the quiescence and proliferation of hematopoietic stem cells. I made the point “Effective mTORC1 negative regulation is essential for keeping the stem cell supply chain working well, at least insofar as hematopoietic stem cells are concerned.” That is, the negative regulation is needed to keep rate of differentiation of hematopoietic stem cells under control so as not to exhaust the pools of such cells.  When the body’s task is regenerating a liver, on the other hand the need is for increased differentiation of stem/progenitor cells to create new hepatocytes, that is activation of the P13K-FRAP/mTOR pathway as described in the above-mentioned publication.  At least, that is my conjecture. 

The same publication mentions activation of the MAPK/ERK pathway.  “–partial hepatectomy induces a rapid but transient activation of mitogenic signal transduction pathways, in particular phosphoinositide 3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)(ref).”  The role of the MAPK/ERK and Notch pathways in the stem cell supply chain are discussed in my recent blog post Niche, Notch and Nudge. MAPK/ERK appears to be very important regulator of Notch activity, and Notch activity in turn is essential for the inter-cellular communication required to form a complete organ.  You can’t get a complicated highly-structured organ like the liver without the cells doing a lot of talking to each other. 

·        Progenitor cell rejuvenation can reverse age-related decline in liver renewal by restoring Notch signaling

The “letter to nature” Rejuvenation of aged progenitor cells by exposure to a young systemic environment discusses the role of Notch signaling with respect to liver progenitor cells.  “The decline in hepatic progenitor cell proliferation owing to the formation of a complex involving cEBP-a and the chromatin remodeling factor brahma (Brm) inhibits the regenerative capacity of aged liver. — The exposure of satellite cells from old mice to young serum enhanced the expression of the Notch ligand (Delta), increased Notch activation, and enhanced proliferation in vitro.  Furthermore, heterochronic parabiosis (a fancy term for pairing up old mice with young mice) increased aged hepatocyte proliferation and restored the cEBPa complex to levels seen in young animals. These results suggest that the age-related decline of progenitor cell activity can be modulated by systemic factors that change with age.” Further, similar pairing of old mice did not result in comparable cell rejuvenation.  “The cEBP- α–Brm protein complex was detected in liver extracts from old isochronic parabionts but not in young isochronic parabionts. Notably, the formation of the cEBP- α –Brm complex was diminished in liver extracts from old heterochronic parabionts. The complex was present at elevated levels in young heterochronic parabionts as compared to young controls. This was also consistent with the modest inhibition of hepatocyte proliferation in young heterochronic parabionts. A young systemic environment restores a young molecular signaling profile to aged progenitor cells in the liver and also appears to enhance their proliferation(ref).”

·        The research that I read leaves me unclear as to the primary actions and locations of telomerase activation and the relationship of telomerase activation to hepatocyte proliferation.   I hypothesize  that one important role of the increased telomerase expression is to accelerate the differentiation of hepatic progenitor cells into new hepatocytes.

The pig-liver researchers stated  “Based on our results, we propose that telomerase activation in proliferating hepatocytes is the main cause for increased telomerase activity in regenerative liver nodules. This conclusion is supported by the significant correlation of telomerase activity to the number of mitotic hepatocytes in our study(ref).” However this statement does not say whether telomerase activation is a cause of the proliferation or a byproduct of it.  And, if there is a causal relationship it does not say what it is. Telomerase activation could possibly a) increase proliferation by promoting the rate of differentiation of hepatic progenitor cells into hepatocytes or b) by the rate of mitosis of such progenitor cells or c) by the rate of mitosis of hepatocytes or by c) delay of senescence of hepatocytes, or by d) any combination of these factors.  To my knowledge telomerase activation is not normally associated with increased mitosis of cells but is known to be able to significantly delay cell senescence and to promote differentiation of stem and progenitor cells, so my guess is that of the above choices all are likely to apply. 

·        Research in liver-related stem cells is recent and partial

The 2005 article Which stem cells for adult liver? demonstrated a lack of concrete knowledge about liver stem cells.  “While hepatocytes can be considered conceptually as unipotent stem cells, the presence of true stem or progenitor cells within adult livers has been largely debated.”  The article goes on to discuss a number of possibilities that might be used for stem cell therapy in livers. Some earlier reports suggested evidence for the presence of hematopoietic stem cells in adult livers(ref).   A November 2008 research report indicates some progress.  “A novel protein marker has been found that identifies rare adult liver stem cells, whose ability to regenerate injured liver tissue has the potential for cell-replacement therapy. For the first time, researchers at the University of Pennsylvania School of Medicine led by Linda Greenbaum, MD, Assistant Professor of Medicine in the Division of Gastroenterology, have demonstrated that cells expressing the marker can differentiate into both liver cells and cells that line the bile duct.”  The marker is the protein Foxl1.

·        Hepatic progenitor and stem cells are possibly implicated in liver cancers

The understanding of cancer stem cells is fairly new and the existence of liver cancer stem cells is an area being investigated actively.  According to a 2009 review paper Liver stem cells and hepatocellular carcinoma.   “Constant proliferation of stem cells is a vital component in liver tissues. In these renewing tissues, mutations will most likely result in expansion of the altered stem cells, perpetuating and increasing the chances of additional mutations and tumor progression. However, many details about hepatocellular cancer stem cells that are important for early detection remain poorly understood”  A September 2009 article reports the isolation of liver cancer stem cells that appear before tumor formation.

·        Telomerase activity may telegraph the possibility of liver cancer

Again, this has been known for a relatively long time.  The 1998 publication Telomerase activity in precancerous hepatic nodules states “These findings suggest that telomerase activation is an early event in large nodule formation in cirrhosis, which may facilitate the action of other factors in the process of carcinogenesis. Telomerase activity in large hepatic nodules is not always indicative of malignant transformation.”  Another publication comes to the conclusions “These results suggest that the induction of hTERT mRNA is an important early event and that its measurement by real-time quantitative RT-PCR is a useful tool to detect premalignant/malignant tendencies in hepatic nodules. However, hTERT gene dosage and c-myc expression are not the main mechanisms regulating hTERT expression in hepatocarcinogenesis.”  And a third 2002 study states “This study shows that hTERT re-expression takes place both in hepatic regeneration occurring in cirrhosis and in the early steps of hepatocarcinogenesis, and involves mainly the beta-splice variant of this molecule. Additional regulatory mechanisms may be required for the expression of the full-length hTERT transcript.” 

The ambivalent attitude to telomerase activation that has been widespread among cancer researchers for many years applies also to some of those concerned with liver regeneration.  The 2007 publication Telomerase activation in liver regeneration and hepatocarcinogenesis: Dr. Jekyll or Mr. Hyde? concludes “At present, it is unclear, whether telomerase activation preserves the non-malignant phenotype and replicative longevity of liver cells or constitutes an early alteration obligatory for an unlimited proliferation and malignant transformation.”  I wonder why this is posed as an either-or choice instead of accepting the overwhelming evidence that both are the case and which one is present depends on circumstances.

·        Progress is being made in decoding the signaling leading to liver cancers

The 2008 paper Hepatocellular cancer arises from loss of transforming growth factor beta signaling adaptor protein embryonic liver fodrin through abnormal angiogenesis suggests a specific mechanism.  “Loss of ELF (embryonic liver fodrin) in the liver leads the cancer formation by deregulated hepatocyte proliferation and stimulation of angiogenesis in early cancers.”  This paper builds on the argument in a 2007 paper Disruption of transforming growth factor-beta signaling through beta-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation.  “Thus, we show that TGF-beta signaling and Smad adaptor ELF suppress human hepatocarcinogenesis, potentially through cyclin D1 deregulation. Loss of ELF could serve as a primary event in progression toward a fully transformed phenotype and could hold promise for new therapeutic approaches in human HCCs.” 

Another 2008 publication CpG island methylator phenotype association with upregulated telomerase activity in hepatocellular carcinoma looks at cancer gene activation in terms of DNA methylation:  “CpG island methylator phenotype (CIMP) involves the targeting of multiple genes by promoter hypermethylation. — We examined the promoter methylation status of 9 genes associated with telomerase activity in 120 HCC, 120 cirrhosis tissues and 10 normal liver tissues by methylation-specific PCR. — CIMP lead to high levels of expression of telomerase activity through the simultaneous inactivation of multiple genes associated with telomerase activity by concordant methylation.” Another interesting study concludes “The results of this study suggest that HBx expression may play a role in hepatocellular carcinogenesis by interfering with telomerase activity during hepatocyte proliferation.”

So, in the light of all of the above the bottom-line guesses to answer my questions are:

1.     What cells are making the telomerase?   I found no direct answers.  I presume both hepatic progenitor cells and hepatocytes are doing so in response to chain of distress signaling present in damaged livers that is only partially understood.

2.     What is the role of the telomerase in liver regeneration?  I hypothesize that there are two important roles: a) enhancing the proliferation potential of hepatocytes and hepatic progenitor cells by allowing a large number of additional population doublings before cell senescence sets in and b) is promoting the health and differentiation of hepatic progenitor cells into hepatocytes.

3.     To what extent is the liver regeneration due to mitosis of existing hepatocytes and to what extent is it due to differentiation of hepatic progenitor cells?   Again I found no direct answer but there seems to be strong evidence that both division of existing cells and differentiation of hepatic progenitor cells play critical roles.

4.     What other stem cells might be involved, such as for renewing the supply of hepatic progenitor cells?  Again there is no simple answer based on experiment.  Several kinds of multipotent stem cells are capable of transforming into hepatocytes and possibly into hepatic progenitor cells.  I conjecture that when liver regeneration is taking place several levels of the stem cell supply chain may be activated.

5.     What kind of signaling is articulating the whole regeneration process?   A number of kinds of signaling are identified in various papers.  Among those of importance that I have discussed before are mTOR, MAPK, and Notch.

6.     What hope does all this offer for longevity?  A great deal, I would say.  First, it appears that the age-related decline in liver regeneration capability can be reversed and that the “heterochronic parabiosis” techniques mentioned above might possibly be adopted so they work in clinical practice.  If they can be made to work they would apply to stem cell proliferation and differentiation in general and could be broadly applied across other organ systems for life extension.  Second, knowledge of the biochemical and genetic pathways active in liver regeneration will probably be  of high relevance in other areas of regenerative medicine.

A research report that appeared a few days ago suggests that birth control pills are having a profound effect on our programmed biological mechanisms for selecting sex partners and for the evolutionary selection of sex and perhaps other human characteristics.

Little stories tell the tale:

·        Before or without the pill it was “Me Tarzan, tough and a good fighter who grabbed you from the other wimpy guys.  You Jane, soft and cuddly strange creature, irresistible to me.  Let’s go have sex again.”  Although Tarzan is a strange animal for her, Jane loves the way he smells and she melts into his arms. Both are responding to biological urges that have kept our species going for millions of years

·        With the pill it is more like “Me Alfred, an insurance lawyer.  You Bridget, a designer of webs for auto dealers.  I found you on Facebook where I liked your little sketches.  We are a lot alike.  Let’s cook a seafood risotto for dinner together and then we can watch Masterpiece Theatre.”  Bridget does not notice anything in particular about how Alfred smells.   They don’t have sex very often but that seems quite fine.  The biological urge is blunted.

·        There is more.  Unbeknownst to Bridget, Alfred has been having a torrid affair with Jennifer, a perky Brazilian pizza-delivery girl who is not on the pill.  There is something about the way that Alfred smells that drives Jennifer to jump into bed with him.  And Alfred cannot resist Jennifer’s allure.  At the moment, Jennifer is pregnant with Alfred’s kid and he is working up courage to tell Bridget about that. The biological urge was working again and has done its thing.

The study which appeared October 6 2009 in the journal Trends in Ecology & Evolution is entitled Does the contraceptive pill alter mate choice in humans?  Basically, birth control pills disrupt the menstrual cycle.  However, the events in the menstrual cycle profoundly affect attractiveness of men for women and women for men and dictate when the sexual urge is mutually maximized.  “Female and male mate choice preferences in humans both vary according to the menstrual cycle. Women prefer more masculine, symmetrical and genetically unrelated men during ovulation compared with other phases of their cycle, and recent evidence suggests that men prefer ovulating women to others.  Such monthly shifts in mate preference have been suggested to bring evolutionary benefits in terms of reproductive success. New evidence is now emerging that taking the oral contraceptive pill might significantly alter both female and male mate choice by removing the mid-cycle change in preferences(ref).”   

The coverage of this study in an October 8 2009 Science Daily article summarizes the situation well.  The study shows “emerging evidence suggesting that contraceptive methods which alter a woman’s natural hormonal cycles may have an underappreciated impact on choice of partners for both women and men and, possibly, reproductive success — – Ovulating women exhibit a preference for more masculine male features, are particularly attracted to men showing dominance and male-male competitiveness and prefer partners that are genetically dissimilar to themselves. This is significant because there is evidence suggesting that genetic similarity between couples might be linked with infertility. Further, some studies have suggested that men detect women’s fertility status, preferring ovulating women in situations where they can compare the attractiveness of different women.” – –  “Dr. Alverne and colleague Dr. Virpi Lumma reviewed and discussed new research supporting the conclusion that use of the pill by women disrupted their variation in mate preferences across their menstrual cycle.

The authors also speculate that the use of oral contraceptives may influence a woman’s ability to attract a mate by reducing attractiveness to men, thereby disrupting her ability to compete with normally cycling women for access to mate.  — Of particular interest is the fact that women taking the pill do not exhibit the ovulation-specific attraction to genetically dissimilar partners(ref).” 

Another study reported last year raises similar issues.  “The contraceptive pill may disrupt women’s natural ability to choose a partner genetically dissimilar to themselves(ref).   ”This study was based on body odors. “Humans choose partners through their body odour and tend to be attracted to those with a dissimilar genetic make-up to themselves, maintaining genetic diversity. Genes in the Major Histocompatibility Complex (MHC), which helps build the proteins involved in the body’s immune response, also play a prominent role in odour through interaction with skin bacteria. In this way these genes also help determine which individuals find us attractive(ref).” 

But those body odors are not fully perceived by women who are on the pill.  “The research team analyzed how the contraceptive pill affects odour preferences. One hundred women were asked to indicate their preferences on six male body odour samples, drawn from 97 volunteer samples, before and after initiating contraceptive pill use. – “The results showed that the preferences of women who began using the contraceptive pill shifted towards men with genetically similar odours(ref).” 

This could be bad news for the stability of a relationship.  “Not only could MHC-similarity in couples lead to fertility problems but it could ultimately lead to the breakdown of relationships when women stop using the contraceptive pill, as odour perception plays a significant role in maintaining attraction to partners(ref).”  (Continuing the little story: When Bridget was finally told about Jennifer being pregnant she stopped having any sex with Alfred and stopped taking the pill.  She soon started experiencing Alfred as disgusting and, despite seeing a marriage counselor and the fact that their relationship had been working exceedingly well, she separated from him after two months.) 

An interesting question is what the implications of contraceptive pill usage might be for offspring and their evolutionary descendents.  Tarzan’s child with Jane and Alfred’s child with Jennifer would seem to continue with the established biological pattern – mating for diversity with male offspring having classical male characteristics and female offspring having classical female ones.  Stereotypical!  (Jane and Tarzan’s daughter got pregnant the first time when she was in high school.)  But what about a child born of Alfred and Bridget or any other couple where the woman is usually on the pill?   Would the lack of selection-for-diversity show up as genetic defects?  “Disturbing a woman’s instinctive attraction to genetically different men could result in difficulties when trying to conceive, an increased risk of miscarriage and long intervals between pregnancies. Passing on a lack of diverse genes to a child could also weaken their immune system(ref).”   

Another interesting question is whether evolutionary selection due to widespread use of the pill will favor people in which traditional male-female odor signaling plays a lesser part in mating and child conception.  That could be a partial explanation for why middle-class people in most advanced countries are getting married and having children much later in life.   Or, is evolutionary selection happening in which Tarzan is getting to be more Jane-like and Jane more Tarzan-like?  Good social arguments can be made that this is happening.  As we go now into the third and fourth generations of women on the pill, these questions could become more relevant. 

And finally, what about decline and blunting of sexual lust, the great traditional builder of populations?  Birth rates are declining in advanced countries where religious norms do not dictate having unlimited numbers of children.  Is the pill a major contributor to stabilization of the world’s population not only by cutting down the number of pregnancies but also by blunting natural sexual attractiveness? 

As a personal note, I have had 5 natural children by 4 wives and have raised an additional 3 as my own.  I fully played ball with the natural biological imperative to reproduce and loved doing so.  However, of those eight children I raised, four have not had their own children so far despite the fact that all are over 30.

The stream of stem cell research seems to be turning into a river with cascades, waterfalls, whirlpools and even stem-cell treatment resorts.  I comment here on just one small part of the river, which is research on generating induced pluripotent stem cells (iPSCs) that are safe to use in human tissues. I also include a “crazy” idea at the end on how to close the loop in the stem cell supply chain, possibly enabling very very long lives.

Three of the key issues being worked on are 1.  making sure that the final iPSC products are free of viral genes or oncogenes or gene translocations, 2. Making sure the iPSCs are free of viral DNA introduced by the process of making them and 3.  raising the efficiency of iPSC production.  I discussed the first two of these issues before.  See June 2009 blog post Update on induced pluripotent stem cells. The original approach to iPSC production was to use a viral vector to insert four genes into the cell to be reverted to iPSC status: Oct4, Sox2, cMyc, and Klf4.  The blog post Rebooting cells and longevity mentions the approach and the alternatives that appeared to be visible back in March 2009, but that is a long time ago as far as iPSC stem cell knowledge is concerned. 

One of the central problems with the original approach was that the DNA of these genes unpredictably integrates itself into the DNA of the iPSCs and cMyc is known to be a potential oncogene. “When Myc is mutated, or overexpressed, the protein doesn’t bind correctly, and often causes cancer(ref).”  If the cells are to be used on humans, traces of those genes are unacceptable.  There is a growing perception that “– residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease(ref).” Virus vectors used for gene insertion for gene insertion are also suspect.  DNA from virus vectors can integrate into the DNA of the iPSC cells, possibly affecting their transcriptional profiles or sometimes even inducing cell death or tumors. What is desired is iPSC cells that are completely free of “footprints” due to how they were created.

One approach to the issue of foreign DNA in iPSC cells is to eliminate insertion of some of the four genes or eliminate their use altogether.  A June 2008 news story tells of four different approaches towards this end being pursued at that time.  Here are some more-recent publications, including one that appeared the day-before-yesterday.

The September 2009 paper Tgfβ Signal Inhibition Cooperates in the Induction of iPSCs and Replaces Sox2 and cMyc offers hope for eliminating use of two of the genes and also addresses the productivity issue.  “iPSC derivation is highly inefficient, and the underlying mechanisms are largely unknown. This low efficiency suggests the existence of additional cooperative factors whose identification is critical for understanding reprogramming. –]. Thus, the identification of compounds that enhance rather than solely replace the function of the reprogramming factors will be of great use. Here, we demonstrate that inhibition of Tgfbβ signaling cooperates in the reprogramming of murine fibroblasts by enabling faster, more efficient induction of iPSCs, whereas activation of Tgfβ signaling blocks reprogramming. In addition to exhibiting a strong cooperative effect, the Tgfβ receptor inhibitor bypasses the requirement for exogenous cMyc or Sox2, highlighting its dual role as a cooperative and replacement factor.”

In a recent blog post I mentioned another September 2009 publication relating to an approach to iPSC induction without introducing any genes into cells at all.  Induction of Stem Cell Gene Expression in Adult Human Fibroblasts without Transgenes.  “Because forced expression of these genes by viral transduction results in transgene integration with unknown and unpredictable potential mutagenic effects, identification of cell culture conditions that can induce endogenous expression of these genes is desirable. — Manipulation of oxygen concentration and FGF2 supplementation can modulate expression of some pluripotency related genes at the transcriptional, translational, and cellular localization level. Changing cell culture condition parameters led to expression of REX1, potentiation of expression of LIN28, translation of OCT4, SOX2, and NANOG, and translocation of these transcription factors to the cell nucleus. We also show that culture conditions affect the in vitro lifespan of dermal fibroblasts, nearly doubling the number of population doublings before the cells reach replicative senescence. Our results suggest that it is possible to induce and manipulate endogenous expression of stem cell genes in somatic cells without genetic manipulation, but this short-term induction may not be sufficient for acquisition of true pluripotency.”  This is work-in-progress but the idea of inducing pluripotency purely through manipulating culture conditions is intriguing.

Another approach to getting rid of the cancer genes from iPSCs was described in my recent blog post Toward a genetic cure for Parkinson’s disease which cites the March 2009 report Breakthrough produces Parkinson’s patient-specific stem cells free of harmful reprogramming genes.  As I said in that post, The approach used by the  researchers is a good example of gene editing.  “In the current method, Whitehead researchers used viruses to transfer the four reprogramming genes and a gene coding for the enzyme Cre into skin cells from Parkinson’s disease patients. The reprogramming genes were bracketed by short DNA sequences, called loxP, which are recognized by the enzyme Cre.  After the skin cells were reprogrammed to iPS cells, the researchers introduced the Cre enzyme into the cells, which removed the DNA between the two loxP sites, thereby deleting the reprogramming genes from the cells. The result is a collection of iPS cells with genomes virtually identical to those of the Parkinson’s disease patients from whom original skin cells came.” 

Yet-another chemical approach for increasing the efficiency of iPSC production is described in the year-old news report Technique for Rapidly Reprogramming Adult Cells Into Stem Cells Published in PLoS Biology.

In September2009, researchers from the University of California, San Diego School of Medicine and the Salk Institute for Biological Studies in La Jolla reported developing “ a safe strategy for reprogramming cells to a pluripotent state without use of viral vectors or genomic insertions.”  The cells produced were pluripotent but-not-quite virgin iPSCs.  “– these induced pluripotent stem cells (iPSCs) are very similar to human embryonic stem cells, yet maintain a “transcriptional signature.” In essence, these cells retain some memory of the donor cells they once were.  “”Working with neural stem cells, we discovered that a single factor can be used to re-program a human cell into a pluripotent state, one with the ability to differentiate into any type of cell in the body” said Muotri (the lead researcher). — “While most of the original genetic memory was erased when the cells were reprogrammed, some were retained,” said Muotri. He added that, in the past, it wasn’t known if this was caused by the use of viral vectors. “By using a footprint-free methodology, we have shown a safe way to generate human iPSCs for clinical purposes and basic research. We’ve also raised an interesting question about what, if any, effect the ‘memory retention’ of these cells might have(ref) .”

Then there is the RepSox approach revealed two days ago.  Of course it was Boston-based researchers (from the Harvard Stem Cell Institute) who come up with that name.  RepSox is a small-molecule compound the researchers discovered that replaces use of the gene Sox2 (thus the name RepSox) when reprogramming cells to iPSC status.  It turns out that RepSox also makes use of the gene c-Myc unnecessary. “– many scientists think the safest approach is to replace the genes altogether with so-called small molecules. In a study published online today in the journal Cell Stem Cell entitled A Small-Molecule Inhibitor of Tgf-β Signaling Replaces Sox2 in Reprogramming by Inducing Nanog, researchers from the Harvard Stem Cell Institute report that a single compound they dubbed RepSox can replace two of the four key reprogramming genes. – “We’re halfway home, and remarkably we got halfway home with just one chemical,” senior author Kevin Eggan, a professor in Harvard’s department of stem cell and regenerative biology, said in a statement. — Now the group will turn its attention to finding other small molecules that could replace the remaining genes – Oct4 and Klf4 – as well, “opening a route to purely chemical programming,” they write(ref).”

Whatever the approach that ultimately turns out to be most successful, researchers are tackling the oncogene, the viral DNA and the productivity problems involved with producing iPSCs that are safe to use with human cells.  These citations are just a small sample of those that already exist and those that can be expected.

A visionary note

A final note of a personal vision.  Suppose, just imagine, that following some of the lines of research described above, a set of small-molecule activators could be identified that selectively causes reversion of a small sub-population of multipotent adult stem cells in-vivo (e.g. hematopoietic stem cells or mesenchymal stem cells) in their niches to return to iPSC status.  Those iPSC cells would then likely respond to niche signaling and differentiate to produce fresh niche-specific multipotent adult stem cells, cells free of the age-related epigenetic burden carried by the older multipotent cells.  What I am imagining is a supplement that closes the loop in the stem cell supply chain which could have a profound longevity-extending effect.  See the blog post The stem cell supply chain – closing the loop for very long lives.  It may sound crazy but if anybody wants to take me on I would make a small even-money bet that we will hear about something like this within the next three years.