Biomedical subjects
R Holliday
Publications and source records attributed to R Holliday.
Further evidence for the rejuvenating effects of the dipeptide L-carnosine on cultured human diploid fibroblasts.
We have confirmed and extended previous results on the beneficial effects of L-carnosine on growth, morphology, and longevity of cultured human fibroblasts, strains MRC-5 and HFF-1. We have shown that late-passage HFF-1 cells retain a juvenile appearance in medium containing 50 mM carnosine, and revert to a senescent phenotype when carnosine is removed. Switching cells between medium with and without carnosine also switches their phenotype from senescent to juvenile, and the reverse. The exact calculation of fibroblast lifespans in population doublings (PDs) depends on the proportion of inoculated cells that attach to their substrate and the final yield of cells in each subculture. We have shown that carnosine does not affect cell attachment, but does increase longevity in PDs. However, the plating efficiency of MRC-5 cells seeded at low density is strongly increased in young and senescent cells by carnosine, as shown by the growth of individual colonies. We have also demonstrated that very late-passage MRC-5 cells (with weekly change of medium without subculture) remain attached to their substrate much longer in medium containing carnosine in comparison to control cultures, and also retain a much more normal phenotype. Carnosine is a naturally occurring dipeptide present at high concentration in a range of human tissues. We suggest it has an important role in cellular homeostasis and maintenance.
Erratum
Colot V, Rossignol J-L. Eukaryotic DNA methylation as an evolutionary device. BioEssays 1999;21:402-411. An error occurred in the above-mentioned article. In the section entitled, "Methylation-associated transcript truncation in Ascobolus and Neurospora," the first line of the second paragraph should have read: Methylation can nevertheless be associated with efficient gene silencing in both fungi. The publisher regrets the error.
The possibility of epigenetic transmission of defects induced by teratogens.
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Causes of aging.
A broad biological approach makes it possible to understand why aging exists and also why different mammalian species have very different maximum longevities. The adult organism is maintained in a functional state by at least ten major mechanisms, which together constitute a substantial proportion of all biological processes. These maintenance mechanisms eventually fail, because the evolved physiological and anatomical design of higher animals is incompatible with continual survival. The life span of each mammalian species depends on the efficiency of maintenance of their cells, tissues, and organs, and there is much evidence that such maintenance is more effective in long-lived species, such as humans, than in short-lived small mammals. It is also evident that there is an inverse relationship between reproductive potential and longevity, which would be expected if available metabolic resources are shared between investment in reproduction and investment in the preservation of the adult body. It is proposed that the eventual failure of maintenance leads to the pathological changes seen in age-associated disease. Although we now have a biological understanding of the aging process, much future research will be needed to uncover the cellular and molecular changes that give rise to age-associated diseases. The major aim of such research is to devise procedures to delay or prevent the onset of these diseases.
Endogenous DNA methylation and epimutagenesis.
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Evidence for gene silencing by endogenous DNA methylation.
Transformed cells can spontaneously silence genes by de novo methylation, and it is generally assumed that this is due to DNA methyltransferase activity. We have tested the alternative hypothesis that gene silencing could be due to the uptake of 5-methyl-dCMP into DNA, via the di- and triphosphonucleotides. 5-Methyl-dCMP would be present in cells from the ongoing repair of DNA. We have isolated a strain of Chinese hamster ovary (CHO) cells, designated HAM-, which spontaneously silences two tested genes at a very high frequency. We have shown that this strain incorporates 5-[3H]methyldeoxycytidine into 5-methylcytosine and thymine in DNA. It also has low 5-methyl-dCMP deaminase activity. Another HAM+ strain has high deaminase activity and a very low frequency of gene silencing. The starting strain, CHO K1, has a phenotype intermediate between HAM- and HAM+.
Gene silencing and endogenous DNA methylation in mammalian cells.
It is known that transformed mammalian cells can spontaneously inactivate genes at low frequency by the de novo methylation of promoter sequences. It is usually assumed that this is due to DNA methyl transferase activity, but an alternative possibility is that 5-methyldCTP is present in these cells and can be directly incorporated into DNA. The ongoing repair of DNA containing 5-methylcytosine will produce 5-methyldeoxycytidine monophosphate (5-methyldCMP), so the question arises whether this can be phosphorylated to 5-methyldCTP. We have tested this using three strains of CHO cells with different levels of 5-methyldCMP deaminase activity. That with the lowest enzyme activity, designated HAM-, has previously been shown to incorporate tritium labelled 5-methyldeoxycytidine into 5-methylcytosine in DNA, with a greater amount of label in thymine. This strain is phenotypically unstable producing cells resistant to bromodeoxyuridine (BrdU) and 6-thioguanine (6-TG) at high frequency. In contrast, the strain with the highest 5-methyldCMP deaminase, designated HAM+, is extremely stable, and the starting strain K1 HAMsl is intermediate between the HAM- and HAM+ phenotypes. We have also shown that human diploid fibroblast strain MRC-5 has a phenotype like HAM+, whereas its SV40 transformed derivative, MRC-5V2 resembles HAM- in having low 5-methyl dCMP deaminase activity, and is phenotypically unstable with regard to 6-TG resistance. It seems that 5-methyldCMP deaminase can be down-regulated in transformed cells, and this can promote de novo methylation by incorporation of 5-methyldCTP derived from 5-methyldCMP.
Is AIDS due to the clonal senescence of CD4 lymphocytes?
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Gene silencing by DNA methylation and dual inheritance in Chinese hamster ovary cells.
Chinese hamster ovary (CHO) cells strain D422, which has one copy of the adenine phosphoribosyl transferase (APRT) gene, were permeabilized by electroporation and treated with 5-methyl deoxycytidine triphosphate. Cells with a silenced APRT gene were selected on 2, 6-diaminopurine. Colonies were isolated and shown to be reactivated to APRT+ by 5-aza-cytidine and by selection in medium containing adenine, aminopterin and thymidine. Genomic DNA was prepared from eight isolates of independent origin and subjected to bisulphite treatment. This deaminates cytosine to uracil in single-stranded DNA but does not deaminate 5-methyl cytosine. PCR, cloning and sequencing revealed the methylation pattern of CpG doublets in the promoter region of the APRT- gene, whereas the active APRT gene had nonmethylated DNA. CHO strain K1, which has two copies of the APRT+ gene, could also be silenced by the same procedure but at a lower frequency. The availability of the 5-methyl dCTP-induced silencing, 5-aza-CR and a standard mutagen, ethyl methane sulphonate, makes it possible to follow concomitantly the inheritance of active, mutant or silenced gene copies. This analysis demonstrates "dual inheritance" at the APRT locus in CHO cells.
Radiology for the surgeon. Case 23. Pancreatic insulinoma (uncinate process)
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Understanding ageing.
A broad biological approach makes it possible to understand why ageing exists and also why different mammalian species have very different maximum longevities. The adult organism is maintained in a functional state by at least ten major mechanisms, which together comprise a substantial proportion of all biological processes. These maintenance mechanisms eventually fail, because the evolved physiological and anatomical design of higher animals is incompatible with continual survival. The lifespan of each mammalian species depends on the efficiency of maintenance of their cells, tissues and organisms, and there is much evidence that such maintenance is more effective in long-lived species, such as man, than in short-lived small mammals. It is also evident that there is an inverse relationship between reproductive potential and longevity, which would be expected if total metabolic resources are shared between investment in reproduction, and investment in the preservation of the adult body. It is proposed that the eventual failure of maintenance leads to the pathological changes seen in age-associated disease. Although we now have a biological understanding of the ageing process, much future research will be needed to uncover the cellular and molecular changes which give rise to age-associated diseases. The major aim of such research is to devise procedures to delay or prevent the onset of these diseases.
Local resection and brachytherapy confined to the lumpectomy site for early breast cancer: a pilot study.
BACKGROUND AND OBJECTIVES: The commonest site of local breast recurrence after breast conservation surgery is the primary tumor bed. We have tested the feasibility of outpatient high dose rate brachytherapy to the primary tumor bed as the only radiation. Our technique relies on the placement of surgical clips to mark the tumor bed. METHODS: Between March 1992 and January 1996, 39 patients with clinical T1 T2 breast cancer were enrolled in this pilot study. The first 13 patients had intraoperative implantation of the breast. The remaining 26 patients had outpatient postoperative implantation under general anesthesia (2 patients) or local anaesthesia (24 patients). High dose rate brachytherapy was given twice daily at least 6 hours apart for a total dose of 37.2 Gy in 10 fractions over 5-7 days. RESULTS: Three patients had mild clinical cellulitis responding to oral antibiotics. One patient had a small sinus in the lumpectomy scar requiring local excision to heal. Four patients developed fat necrosis at the lumpectomy site at 4 (1 patient), 13 (1 patient), and 18 months (2 patients) post radiotherapy. Patient rated satisfaction with treatment was high. At a median followup of 20 months, one infield local recurrence has been salvaged by wider resection and postoperative conventional external beam radiation. CONCLUSIONS: Except for fat necrosis, which may be associated with this technique, complications have been minimal. Outpatient implantation under local anesthesia is feasible. Longer followup is required to establish the local control rates.
Evolution: the dialogue between life and death.
Organisms have the ability to harness energy from the environment to create order and to reproduce. From early error-prone systems natural selection acted to produce present day organisms with high accuracy in the synthesis of macromolecules. The environment imposes strict limits on reproduction, so evolution is always accompanied by the discarding of a large proportion of the less fit cells, or organisms. Sexual reproduction depends on an immortal germline and a soma which may be immortal or mortal. Higher animals living in hazardous environments have evolved aging and death of the soma for the benefit of the ongoing germline.
Endless quest.
The replication of linear chromosome DNA by DNA polymerase leads to the loss of terminal sequences, in the absence of a special mechanism to maintain ends or telomeres. This mechanism is known to consist of short terminal repeats and the enzyme telomerase, which contains RNA complementary to the DNA repeats. There is evidence that telomeric DNA continually decreases in size in the absence of telomerase, and this is followed by cellular senescence. Immortalisation of somatic cells is accompanied, at least in some cases, by acquisition of telomerase activity. The cloning of DNA coding for the RNA component of telomerase has opened up some new experimental approaches, including the study of telomerases with mutant RNA. The telomere theory of cellular senescence appears to provide a molecular basis for the 'Hayflick limit' to human fibroblast growth. However the telomeres and behaviour of primary mouse cells are anomolous, and many immortalised human cell lines lack normal telomerase activity. These exceptions are not easily accommodated in the telomere theory.
The urgency of research on ageing.
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Inhibition of the growth of transformed and neoplastic cells by the dipeptide carnosine.
Human diploid fibroblasts growth normally in medium containing physiological concentrations of the naturally occurring dipeptide carnosine (beta-alanyl-L-histidine). These concentrations are cytotoxic to transformed and neoplastic cells lines in modified Eagle medium (MEM), whereas these cells grow vigorously in Dulbecco's modified Eagle medium (DMEM) containing carnosine. This difference is due to the presence of 1 mM sodium pyruvate in DMEM. Seven human cell lines and two rodent cell lines were tested and all are strongly inhibited by carnosine in the absence of pyruvate. Experiments with HeLa cells show that anserine is similar to carnosine, but D-carnosine and homocarnosine are without effect. Also, the non-essential amino acids alanine and glutamic acid contribute to the effect of pyruvate in preventing carnosine toxicity, and oxaloacetate and alpha-ketoglutarate can substitute for pyruvate. We have used mixtures of normal MRC-5 fibroblasts and HeLa cells to demonstrate that 20 mM carnosine can selectively eliminate the tumour cells. This has obvious implications which might be exploited in in vivo and in vitro studies. Carnosine is known to react strongly with aldehyde and keto groups of sugars by Amadori reaction, and we propose that it depletes certain glycolysis intermediates. It is well known that tumour cells are more dependent on glycolysis than normal cells. A reduction of glycolysis intermediates by carnosine may deplete their energy supply, but this effect is totally reversed by pyruvate.
The evolution of human longevity.
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