特別寄稿
IN MEMORY OF DR. ROBIN HOLLIDAY, HIS LABORATORY IN MID-NINETEEN EIGHTIES, AND TESTING DNA METHYLATION THEORY OF IN-VITRO CELLULAR AGING
2015 年 34 巻 2 号 p. 133-138
詳細
2015 年 34 巻 2 号 p. 133-138
Robin Holliday, a noted British philosopher and a sculptor, born in 1932 died in 2014. After his remarkable contribution in nineteen sixties and seventies to modern molecular biology through his presentation of a genetic recombination model, i.e., Holliday model, and his proposal of epigenetic control mechanisms based on DNA methylation, he poured in nineteen eighties his scientific interests in the mechanisms of aging, particularly in solving the problem of in-vitro limited proliferative lifespan of cultured human diploid cells (HDCs). Two major findings obtained during that time in his laboratory were that cell’s memory of proliferative life span can be modified with a pulse treatment of DNA-methyl transferase inhibitors, 5-azacytidine and 5-azadeoxycytidine, and that SV40-infected-, but not immortalized, HDCs maintain their level of DNA methylation until the end of their proliferative life span. It was shown with these two lines of findings that the drift of DNA-methylation level is influential, but not essential determinant of the limitation of proliferative potential. These studies underlay further studies of Hayflick limit in nineteen nineties including the discovery of telomerase participation.
Robin Holliday, PhD, Fellow of the Royal Society and of the Australian Academy of Science, a noted British philosopher and a sculptor, born 6 November 1932 died 9 April 2014. As one of those who were given the opportunity to join his research group, I want to extend my deep condolence to Mrs. Holliday and to all who know him.
Hereafter, research works performed in mid eighties in his group are retrospectively reviewed for future advances of related researches together with my warm memories of Dr. Robin Holliday and his group members in those days when we got together in Mill Hill.
For general introduction to Dr. Holliday, readers of this article are suggested to refer to obituaries presented by Leonard Hayflick1), Bill Holloman2), Penny Jeggo and Fotini Gounari3), and Tom Kirkwood4).
Needless to mention, the first big accomplishment of Dr. Holliday was his presentation of a structural model of homologous recombination which has been materialized and now known as Holliday junction5).
His second one, partly with John Pugh, as big as or even bigger than the first one, was his proposal of epigenetic control mechanisms based on DNA methylation6,7). With this proposal of his, a clear materialistic image in molecular terms was first drawn to explain epigenetic phenomena, which triggered and stimulated the earth-shaking progress of modern human biology including not only developmental biology and gerontology, but behavioral and neuro-sciences8). These two monumental accomplishments were attained during nineteen sixties to seventies in his thirty to forty years of age.
In nineteen eighties, Dr. Robin Holliday’s interests were intensively poured into the mechanisms of aging. In those days, the fact that the proliferative potential of cultured normal somatic human diploid cells (HDCs hereafter) is limited had already been established since early sixties by Leonard Hayflick and Paul Moorhead9), and by Leonard Hayflick10). By eighties, evidence had already been accumulated suggesting that the finite potential of cellular proliferation is related to the finite potential of body life. However, the molecular mechanisms of the limited in-vitro proliferative potential, i.e., Hayflick limit, remained yet to be known, as one of representing biological problems of the decade.
In 1983 Vincent Wilson and Peter Jones reported that DNA methylation decreases toward the end of in-vitro proliferative life span of HDCs and other normal diploid cells, while it did not in immortalized cells11).
Thus, the DNA methylation theory of limited life span, as well as Peter Medawar’s mutation theory and Leslie Orgel’s error catastrophe theory of aging, emerged as an attractive theory that could possibly explain the mechanism of Hayflick limit.
From late seventies to early eighties I was a Japanese government-employed research scientist, who was back from Len Hayflick’s laboratory at Stanford, again to Institute of Medical Science, University of Tokyo. There I was genealogically analyzing family trees of cultured rodent cells using microscopic time-laps cinematography during the course of their in-vitro senescence and immortalization12). During that period of time in addition, I was partly engaged, together with Makoto Goto and Toshiharu Ishii, in the distribution of Werner syndrome cells within scientific community, and also trying to obtain immortalized Werner cells by SV40 transformation.
It was December 1982 when I first saw Dr. Holliday in Tokyo in an international meeting on premature aging organized by Yoshisada Fujiwara, Darrel Salk and George Martin. In that meeting he presented the culture history and cellular characteristics of SV40 transformed- and immortalized Werner syndrome cells13).
My first impression of Dr. Holliday was as if I had already seen him, or at least a man with similar hair style, in a photograph of the most famous British writer which appeared somewhere in one of my high school textbooks, a warm-hearted man with invincible passion, while sometimes with shy smile.
With an idea to test the DNA methylation theory of aging, and an additional research subject in the field of biomedical ethics on the use of human tissues and cells in research and industry in mind, I applied for an IARC scholarship and for a Japanese government scholarship, both of which accepted my application to stay in Dr. Holliday’s laboratory, and he kindly accepted my stay. Thus, I stayed in his laboratory in National Institute for Medical Research in London as a visiting scientist from February 1985 to April 86.
In February 1985, I arrived at the National Institute of Medical Research, London. There, I noted that all members of Genetic Division called Dr. Holliday as Robin, and so did I, as I do also hereafter.
In those days there were more than fifteen members in Genetic Division. Sorry for that I might not remember all, but among those who I remember were Geoff Banks, Ian Buchanan, Fotini Gounari, Lily Huschtscha, Penny Jeggo, Khash Khazaie, Zhores Medvedev and Rita Medvedeva, Robert Rosenberger, Steve Sedgwick, Ad Spanos, Tony Stevens and Sue Taylor. Jackie Hunter and Farooq Malik joined after I arrived.
There were three-four small groups each with respective leaders persuing their respective missions, while twice a day most members of the division used to get together in a small attic to take tea break when they made arrangements and enjoyed short chattering. The attic room was on the top floor of the tall NIMR building which stood on the top of Mill Hill, being simple except that there was a glass breeding box in which a couple of walking sticks (Phasmatodea species) were taken care by Robin feeding them with blackberry leaves.
In the first couple of conversations, Robin gave me suggestions for taking meals, since I lived alone for a month or so before my family’s arrival. He kindly suggested me to take a dessert after meals. Now I was in the UK! A meal did not finish without dessert!
While chattering, I found Robin’s interests were not only in genetics and aging. He appeared to appreciate me more for my previous works on echinoderm phylogeny than for my previous works on cellular aging. Perhaps, he might appreciate me more if I played the violin. I later learned that he was not only a noted scientist but also a noted sculptor as well.
To my understanding there were two major streams of research running in Genetic Division in those days, one from molecular biochemical approaches toward genetic and epigenetic mechanisms and the other in model-building and testing-theory ways of research mainly oriented to aging problems.
In the former stream were Geoff Banks, Ad Spanos and others studying yeast genetics, and Penny Jeggo, Fotini Gounari and others studying DNA methylation mechanisms using CHO cells. In some studies they formed a large group together.
In the latter, Robin was trying to evaluate DNA methylation theory of aging14). Particularly, he was trying to intervene HDCs life span with inhibitors of DNA methyltransferase.
There, I was given the opportunity to set up a second small group in the latter stream to test DNA methylation theory from the transformation point of view.
In addition, Zhores Medvedef was always in his small office with his handy manual typewriter and Rita Medvedeva at a comfortable corner of laboratory, and so on.
With my apology to skip those works lead by Geoff, Penny and others, except for mentioning the invaluable one in that Fotini and all put their effort together to obtain single temperature sensitive CHO mutant clone of DNA methylation out from seven thousand colonies of CHO cells and to characterize it15), I recall only the latter approaches hereafter.
Robin’s approach in mid eighties was to test theories of in-vitro senescence by intervening cultured HDC with 5-azacytidine (5-aza-CR) or 5-azadeoxycytidine (5-aza-CdR), potent inhibitors of DNA methylation. If helped by Lily and others sometime, many times I saw Robin himself in front of a laboratory safety cabinet handling cell cultures.
He found that a single pulse treatment of HDCs with either 5-aza-CR or 5-aza-CdR resulted in significantly earlier entrance of cultured MRC-5 cells into senescent phase, as if the cells remembered the treatment16). This was in sharp contrast to the little effects shown by DNA-damaging agents, such as ethylmethanesulfonate(EMS) on the lifespan of HDCs. These results supported the idea that, with these drugs, methyl residues at some sites of the parent cell DNA were lost in their sibling cells, while maintenance methylase did not transfer methylation patterns correctly to their sibling cells at the time of DNA replication, and that the modified pattern of DNA methylation, thus created, was then inherited epigenetically to the successor cells showing early entrance into senescent phase.
It was known that, upon infection by SV40, normal HDCs show apparently transformed morphology but only seldomly transform themselves into immortal cells. Therefore, the acquisition of transformed morphology and the acquisition of indefinitely proliferative potential appeared to be independent matters from each other, although the relationship between the two was not clearly understood in those days. Therefore, I thought it worth testing if the DNA methylation level decreases, or it is maintained, during in vitro proliferative life span of SV40-infected, but not immortalized, HDCs.
Upon my arrival, Robin made quick arrangements for me to work with Jackie Hunter and Farooq Malik so that an experimental platform starting with the initiation of MRC-5 cell cultures, SV40 infection, DNA extraction from serially passaged cell cultures, and the HPLC analyses of DNA methylation level was quickly set-up. Jackie, Farooq and I worked hard for some time.
The results showed that the DNA methylation level of SV40-transformed cells did not significantly decrease during their limited lifespan, an indication that there should be other factor(s) than the drift of DNA methylation level that limit(s) the proliferative potential17,18).
Although these results did not devaluate the participation of DNA methylation in aging, it became clear that there is, or are, other essential factor(s) limiting in-vitro life span than those controlling DNA methylation level.
Zhores Medvedev’s activity could not be locked within the framework of Genetic Division, covering such a wide range of science and history including gerontology, radiation biology, aids study and Russian modern history.
It should only be mentioned here that Zhores and Rita pursued studies on hepatocyte polyploidization in respects of cancer and aging19).
As to the additional line of study of myself regarding the ethics on using human tissues and cells for science, technology and industry, I was given the opportunity to see Prof. Ian Kennedy, at the University of London, to join in a meeting on ethics he organized, and to see several persons with mutual interests in and around London. My work on that line was developed further back in Japan and was published many years later20).
Only two years after I left Genetic Division in 1986, Robin left NIMR and moved to Sydney where he maintained his scientific activity obtaining a senior research position in CSIRO. Sorry for that I missed the chance of visiting him there, I had been informed that he was not only active as a scientist, but also as a sculptor, and as a husband of his second wife Lily with whom they were given birth of a daughter Mira.
The life style as he chose with Lily might be rare or unthinkable in old days when an average human life span was only fifty to sixty. However, it may reasonably be anticipated that human life style changes due to the significant extension of life span.
I propose here that this life style of Robin to be referred to as Holliday Model of Extended Senior Life.
In nineteen nineties, there were some monumental advances obtained in the field of aging research that were underlain by those obtained in eighties.
In 1990 Harley and others reported that decreasing telomere lengths is associated with in-vitro cellular proliferative lifespan, suggesting the participation of telomerase in Hayflick limit21). In 1998, Bodner and others confirmed the participation by the introduction of telomerase gene, which extended the proliferative life span22).
In 1996, the gene responsible for the premature aging in Werner syndrome, another focus of interests in aging research, was cloned by Yu and others23).
As to DNA methylation study, a vast amount of sequence data has become available by the development of DNA sequencing technology, through which a new area of DNA methylation study, which is now called methylome24), has arose in which the mode and change of DNA methylation pattern are subject to detailed studies on individual cells, tissues and organs in regards to their development, differentiation and aging25).
Perhaps the time may come again soon when we need another Robin Holliday to draw a visual hypothetical image of aging with which we could sort out these massive amount of complex experimental data.
In 1985 to 86 when I stayed in London, life in suburban London was safe and splendid as it has been so to the presence. Together with my family, I enjoyed weekend driving tours all around England, once in Scotland and once in Wales. All members of my family, including three schoolboys, got invaluable experiences there. Together with my family I was kindly entertained by a number of members of Genetic Division. Among them, a home party at my residence became an unforgettable one. When I invited Robin and others to my residence, Lily Huschtscha told me that her farther was coming to London from Australia, and would come with her. Of course, I told her that he was very welcome. Robin came to my house with a tie around his neck! It was only later when I knew that it had been the first time when Robin met Lily’s farther, and that Lily married Robin, to establish their home in Australia, and to give birth to their daughter Mira.
After we returned back to Japan in 1986, Robin came to Japan and stayed with us in my house in Fujisawa, and made a trip to Hakone Hot Springs in 1987, when I saw him for the last time. He presented us with a pot of orchid plant which has been taken care by Kiyoko Matsumura, and continues to produce purple-white flowers every year.
In addition to my memory of Robin, I want to mention my warm memory of Geoff Banks who was about the same age as me, but passed away in the midst of his active research career after I returned from London.
Robin Holliday in his laboratory in one day 198626).
My acknowledgements go to Zhores Medvedev, Lily Huschtscha-Holliday, Jackie Hunter, Farooq Malik and all who kindly accepted me, helped me, and allowed me to spend such a splendid time of pursuing research science together.
