Biomedical subjects
Publications and source records attributed to V M Craddock.
Newborn rats and adult animals in which liver cell replication had been induced by partial hepatectomy were injected with 5-bromodeoxyuridine (BUdR), and were kept without further treatment for the remainder of their life span. No evidence for carcinogenicity of BUdR was obtained, but a dose-dependent shortening of the life span was observed after administration to neonatal animals.
To study the roles of DNA polymerases alpha and beta during replication and repair of damaged DNA, use was made of the fact that during chronic treatment with carcinogens, replication and repair do not necessarily follow the same time sequence. Early cell damage and restorative hyperplasia cause a transient wave of DNA synthesis, while repair replication might be expected to continue throughout the period of treatment with the carcinogen. N-acetylaminofluorene (AAF) was fed in the diet for periods of up to 35 weeks, and at intervals during the feeding period measurements were made of DNA synthesis in vivo, and of DNA polymerases alpha and beta as assayed in vitro after fractionation. The activity of polymerase alpha increased and decreased with the transient early wave of DNA synthesis. Polymerase beta showed an initial rapid increase in activity which peaked before the increase in DNA synthesis, and then decreased. The decrease in activity may be due to the fact that, although AAF continues to be fed in the diet, the foci and nodules which develop no longer metabolise the carcinogen to a form which damages DNA. Thus replication occurs in the nodules while DNA damage and repair occur in the surrounding non-neoplastic liver. With the rapid growth of nodules there is overall an increase in neoplastic tissue, a relative decrease in nonneoplastic tissue, and thus a relative decrease in DNA damage, repair, and induction of polymerase beta. Histological examination showed that by 35 weeks the conversion to neoplasia was virtually complete. These results support the concept that polymerase alpha functions in de novo replication of DNA, and is induced during cell replication, while polymerase beta functions in repair replication, and increases in activity during chronic damage to DNA. Whether it is induced by treatment with carcinogens depends on the duration of treatment, and on other processes (e.g. metabolism of the carcinogen) which take place during the development of malignancy.
A single injection of dimethylnitrosamine (DMN) given to adult rats induces hepatocellular carcinoma if given during the period of restorative hyperplasia following partial hepatectomy. The effect of DMN on the sequence of biochemical events in regenerating liver is therefore of interest. As carcinogenesis is likely to involve a change in control of gene expression, and as evidence suggests that non-histone chromosomal proteins (NHP) are involved in gene control, the effect of DMN on synthesis of NHP in regenerating liver was studied. A well-defined group of NHP, the high mobility group (HMG), known to be specifically associated with DNA active in transcription, were investigated. Partial hepatectomy was found to cause a large increase in incorporation of [3H]lysine into HMG proteins; this did not occur immediately, but was apparent at the time of DNA synthesis and of mitosis. DMN did not alter the relative amounts of different HMG proteins in relation to histone H1, but the carcinogen caused a considerable reduction in incorporation of [3H]lysine. This decreased synthesis of the proteins which may function as gene derepressors may well be relevant in carcinogenesis.
It has often been suggested that the high molecular weight DNA polymerase alpha of eukaryotes plays a role in de novo replication of DNA, while the low molecular weight polymerase beta is involved in repair replication. Previous studies have shown that when diethylnitrosamine is fed in the diet to rats it causes after a few weeks an increase in de novo replication of DNA, which then returns to normal values. In contrast, repair replication may be expected to continue throughout the feeding period. Study of DNA polymerase activity in livers of animals during carcinogenesis showed that an increase in polymerase alpha occurred at the time of increased de novo replication, while there was a gradual increase in polymerase beta during the time diethylnitrosamine was present in the diet. When diethylnitrosamine treatment was stopped, there was a rapid drop in polymerase beta activity. These results support the view that the polymerase alpha is involved in DNA replication, that the polymerase beta functions in repair replication, and that the beta enzyme can be induced by chronic damage to DNA.
The possibility that carcinogens may affect methylase-mediated methylation of replicating DNA was investigated. A system eminently suitable for this purpose is liver regenerating after partial hepatectomy, as one injection of dimethylnitrosamine (DMN) given during the ensuing period of increased DNA synthesis induces hepatocellular carcinoma. Methylation of DNA by DNA methylase normally occurs only in proportion to DNA synthesis. Therefore simultaneous measurements were made of synthesis (incorporation of [14C]adenine into DNA adenine, or of d[5-3H]cytidine into DNA cytosine), and of methylation (incorporation of [methyl-3H]methionine into 5-methylcytosine of DNA) in liver regenerating after partial hepatectomy. After treatment with DMN, the ratio of methylation: synthesis remained within the normal range. Methyl methanesulphonate (MMS), a compound which damages DNA in regenerating liver in a similar but not identical way to DMN and which does not induce tumors in liver even when given after partial hepatectomy, caused an increase in methylation in relation to synthesis. These experiments therefore do not support the view that altered DNA methylase activity is involved in carcinogenesis.
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To relate repair of lesions in DNA with carcinogenesis, it is necessary to study repair in the intact animal under conditions which will and will not induce cancer. The methods used to study the replications stage of repair in isolated cells are not suitable for use in the intact animal. A method is presented which depends on the fact that during cell replication the nuclei enlarge, and may be separated from nuclei of non-replicating cells by rate zonal centrifugation on a sucrose gradient in a zonal rotor. Thus incorporation of [3H]thymidine as a result of de novo synthesis of DNA in replicating nuclei can be separated from incorporation due to repair synthesis in non-replicating nuclei. Treatment of animals with dimethylnitrosamine or with methyl methanesulphonate produced a "repair-type" profile, which contrasted with that given by liver nuclei from untreated animals, or from animals in which DNA synthesis had been reduced by treatment with cycloheximide, a compound which is not known to cause direct damage to DNA. Evidence is presented which suggests that the method is a rapid sensitive test for the occurrence of repair replication of some kind in the liver of the intact animal.
A single treatment with dimethylnitrosamine (DMN) but not with methyl methanesulphonate (MMS) induces liver cell carcinoma if given during the period of restorative hyperplasia following partial hepatectomy, a higher incidence of tumours being induced if the carcinogen is given during the period of DNA synthesis (24 h after the operation) than if given early in the prereplicative stage (at 6 h). To study the effect of treatment with DMN and with MMS on the regenerating liver, DNA replication was measured in vivo in partially hepatectomised animals treated with the methylating agents, and DNA polymerase activity was assayed in vitro.
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A single injection of dimethylnitrosamine (DMN), 12.0-15.6 mg-kg, given to 100 g female rats 24 h after partial hepatectomy, induced hepatocellular carcinoma. No animals receiving DMN without partial hepatectomy developed liver carcinomas. Previous evidence had suggested that the incidence of tumours was highest when DMN was administered during the wave of DNA replication which follows partial hepatectomy. The present experiments made this suggestive evidence statistically significant. A single treatment with diethylnitrosamine (DEN) induced liver cell cancer when given to intact or to partially hepatectomised rats. No tumors developed when another alkylating carcinogen, methyl methanesulphonate (MMS), was administered after partial hepatectomy. The significance of these results in relation to the mechanism of initiation of carcinogenesis is discussed.
Experiments were carried out to determine whether replication of alkylated DNA could be involved in the initiation of hepatocellular carcinoma which results from a single administration of dimethylnitrosamine (DMN) given after partial hepatectomy. The incidence of tumours is higher when DMN is given during the wave of DNA synthesis induced by the operation than when given in the early prereplicative stage. Therefore the alkylation of DNA in the regenerating liver by DMN given at these times and the effect of DMN on DNA synthesis were investigated. The extent, duration and pattern of alkylation of DNA, including the formation of 0-6-methylguanine, were similar whether DMN was given in the early pre-replicative stage (6 h after the operation) or during the period of DNA synthesis (at 24 h). DMN given a 6 h very greatly reduced the wave of DNA replication which would otherwise have ensued. When given at 24 h, by which time DNA synthesis was already taking place, DMN reduced the rate of incorporation of (-3H)thymidine after 1-2 h delay. However, in neither case was DNA synthesis reduced to the level occurring in normal intact liver. Treatment with diethylnitrosamine (DEN) at 6 h or at 24 h had a similar effect to DMN on the wave of DNA replication induced by partial hepatectomy. Methyl methanesulphonate (MMS given in the early pre-replicative stage delayed the wave of DNA synthesis by about 8 h, but when it did take place the extent of synthesis was as great as in untreated animals. When given during the period of DNA replication, MMS rapidly reduced the rate of synthesis. As in the case of the nitrosamines, synthesis was not reduced to the level occuring in normal intact animals. The difference from the nitrosamines lies in the nature of the alkylated bases formed in DNA. The fact that a single treatment with DMN induces cancer in partially hepatectomised animals but not in intact adult animals is not considered to be due to a gross difference in the nature of the alkylation of DNA. The experiments described support the concept that replication of DNA containing bases which are likely to mispair during replication may be necessary to 'fix' the lesion and thus cause a permanent inheritable change in the genetic material.
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A single treatment of adult animals with the potent carcinogen NMU was known to induce tumours in a wide variety of organs, with the notable exception of liver. Administration of NMU after partial hepatectomy gave rise to the first liver cell adenomata ever observed in rats due to this carcinogen. The tumours were induced when NMU was given during the period of increased DNA synthesis but not when given early in the pre-replicative period. Although tumours were induced in other organs, the incidence of these did not correlate with the timing of NMU administration. It is suggested that replication of damaged DNA may be a relevant event in carcinogenesis.
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