Alexander A. Morley

Cytokinesis-block micronucleus method in human lymphocytes : effect of in vivo ageing and low dose X-irradiation

The cytokinesis-block micronucleus technique was developed to overcome the kinetic problems inherent in the use of human lymphocytes for micronucleus assays. Using this technique the number of spontaneous micronuclei in lymphocytes from 42 individuals aged between 20 and 85 years was studied and was found to increase at a rate of 4.3% per year. Comparison with the results obtained with the conventional micronucleus assay confirmed that the conventional method markedly underestimates this age effect. The sensitivity of the cytokinesis-block method was determined by studying the effect of low-dose (less than 50 rad) X-irradiation. The results indicated that the dose-response was linear and a single in vitro exposure to 5 rad of X-rays could be unequivocally detected. We concluded that the cytokinesis-block micronucleus method is more sensitive and precise than the conventional micronucleus method and classical metaphase analysis, and that it will be of value for detecting chromosome damage induced in vivo by genotoxic agents.

Monoclonality in B cell lymphoma detected in paraffin wax embedded sections using the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to develop a simple technique for detecting monoclonality at the DNA level in B lymphocyte populations in formalin fixed, paraffin wax embedded material. Sections were dewaxed and dehydrated, and the DNA was extracted by boiling in water for 45 minutes. A semi-nested PCR was performed to amplify the V-D-J region of the immunoglobulin heavy chain gene. The product was electrophoresed and viewed under ultraviolet light after ethidium bromide staining. Specimens from 26 B cell lymphomas produced a monoclonal band in 24 cases and no amplification in two cases; monoclonality was specific for this disorder. Specimens from seven T cell lymphomas produced no amplification; specimens from nine reactive nodes produced a broad smear of polyclonal material; and specimens from 12 cases of carcinoma produced either no amplification or polyclonal material. As detection of monoclonality is strongly suggestive of neoplastic disease, this technique is likely to be of value in routine diagnosis, because of its speed, simplicity, and applicability to fixed, embedded material.

Outcome prediction in childhood acute lymphoblastic leukaemia by molecular quantification of residual disease at the end of induction

Methods to detect and quantify minimal residual disease (MRD) after chemotherapy for acute lymphoblastic leukaemia (ALL) could improve treatment by identifying patients who need more or less intensive therapy. We have used a clone-specific polymerase chain reaction to detect rearranged immunoglobulin heavy-chain gene from the leukaemic clone, and quantified the clone by limiting dilution analysis. MRD was successfully quantified, by extracting DNA from marrow slides, from 88 of 181 children with ALL, who had total leucocyte counts below 100 x 10(9)/L at presentation and were enrolled in two clinical trials, in 1980-84 and 1985-89. Leukaemia was detected in the first remission marrow of 38 patients, in amounts between 6.7 x 10(-2) and 9.9 x 10(-7) cells; 26 of these patients relapsed. Of 50 patients with no MRD detected, despite study of 522-496,000 genomes, only 6 relapsed. The association between MRD detection and outcome was significant for patients in each trial. In the first trial, patients relapsed at all levels of detected MRD, whereas in the later trial, in which treatment was more intensive and results were better, the extent of MRD was closely related to the probability of relapse (5 of 5 patients with > 10(-3) MRD, 4 of 10 with 10(-3) to 2 x 10(-5), 0 of 3 with levels below 2 x 10(-5), and 2 of 26 with no MRD detected). Early quantification of leukaemic cells after chemotherapy may be a successful strategy for predicting outcome and hence individualizing treatment in childhood ALL, because the results indicate both in-vivo drug sensitivity of the leukaemia and the number of leukaemic cells that remain to be killed by post-induction therapy.

Development of a highly sensitive assay, based on the polymerase chain reaction, for rare B-lymphocyte clones in a polyclonal population.

A method has been developed to use the polymerase chain reaction to amplify and sequence the chain determining region 3 (CDR3) of the human immunoglobulin heavy‐chain gene, and to use the sequence as a marker for rare neoplastic B lymphocytes. Consensus primers for the Variable and Joining regions of the gene were constructed and shown to enable efficient amplification, directed cloning, and sequencing of CDR3. Using leukaemic cell line PFMC as a test system, CDR3 was sequenced, specific primers synthesized, and PFMC DNA was detected down to a dilution of 1:1300 in DNA from normal lymphocytes. This strategy should be useful for monitoring therapy and detecting early disease relapse in B lymphoproliferative disease.

The effect of donor age on spontaneous and induced micronuclei

The spontaneous micronucleus yield in lymphocyte cultures from healthy donors aged 0-82 years was estimated at 72 h and 96 h of culture. At both 72 and 96 h there was a positive correlation of micronucleus expression with increasing age (p less than 0.001), with an approximately 4-fold increase in micronuclei in cultures from 80-year-old donors when compared to cultures from newborn donors. Since there is some evidence that the effect of DNA-damaging agents may increase with age, lymphocytes from individuals of various ages were exposed to X-rays and mitomycin C and micronuclei were scored after 72 and 96 h of culture. Micronucleus formation after exposure to these agents was, however, decreased in cells from elderly individuals, most likely due to kinetic differences between the lymphocytes of old and young individuals.

Mutation frequency in human lymphocytes increases with age.

Several theories of ageing predict that somatic mutations should increase with age. This prediction was tested for human lymphocytes using a recently developed clonal technique for enumeration of mutations, and an increase of 1.6% per year in mutations with age was detected.

Exposure of human lymphocytes to ionizing radiation reduces mutagenesis by subsequent ionizing radiation

The effect of prior incubation with [3H]thymidine on survival and mutagenesis after X-irradiation of human lymphocytes was studied by incubating lymphocytes with 0.001-1.0 mu Ci/ml [3H]thymidine for 6 h at 37 degrees C and then irradiating with 150 or 300 rad. Survival was measured using lymphocyte cloning and mutagenesis was measured using 6-thioguanine selection to detect clones mutated at the hypoxanthine phosphoribosyltransferase locus. [3H]Thymidine alone had no effect on survival or mutagenesis and X-radiation alone produced the expected decrease in survival and increase in mutations. [3H]Thymidine prior to X-radiation had no effect on lethality of X-radiation but at concentrations of 0.1 and 1.0 mu Ci/ml produced a significant decrease in the number of mutations induced after both 150 and 300 rad. The results suggest that ionizing radiation, produced by disintegration of 3H, reduces the mutagenic effect of a subsequent exposure to ionizing radiation by induction of a system which prevents or repairs a restricted class of radiation damage.

The Linear No-Threshold Model does not Hold for Low-Dose Ionizing Radiation

Abstract Hooker, A. M., Bhat, M., Day, T. K., Lane, J. M., Swinburne, S. J., Morley, A. A. and Sykes, P. J. The Linear No-Threshold Model does not Hold for Low-Dose Ionizing Radiation. Radiat. Res. 162, 447–452 (2004). Almost all of the data on the biological effects of ionizing radiation come from studies of high doses. However, the human population is unlikely to be exposed to such doses. Regulatory limits for radiation exposure are based on the linear no-threshold model, which predicts that the relationship between biological effects and radiation dose is linear, and that any dose has some effect. Chromosomal changes are an important effect of ionizing radiation because of their role in carcinogenesis. Here we exposed pKZ1 mice to single whole-body X-radiation doses as low as 1 μGy. We observed three different phases of response: (1) an induction of inversions at ultra-low doses, (2) a reduction below endogenous inversion frequency at low doses, and (3) an induction of inversions again at higher doses. These results do not fit a linear no-threshold model, and they may have implications for the way in which regulatory standards are presently set and for understanding radiation effects.

Effect of dietary restriction on in vivo somatic mutation in mice.

Dietary restriction is the one experimental variable which has been shown reproducibly to retard ageing. The effect of dietary restriction on in vivo mutation was studied in mice by feeding experimental mice 60% of the amount eaten by control mice and measuring mutations in lymphocytes at the hypoxanthine phosphoribosyl transferase locus at 4 weeks, 6 months and 12 months of age. Dietary restriction markedly decreased the age-associated accumulation of mutations observed in control mice, which suggests that somatic mutation is involved in the ageing process and that the majority of in vivo mutations result from dietary factors.

Mutations in human lymphocytes studied by an HLA selection system

Human lymphocytes mutated at the HLA-A2 or HLA-A3 alleles were enumerated and studied by primary selection using antibody and complement, followed by limiting dilution cloning and secondary selection using immunofluorescence or antibody and complement. The geometric mean frequency of in vivo mutant lymphocytes was 3.08 X 10(-5) for the HLA-A2 allele and 4.68 X 10(-6) for the HLA-A3 allele. Mutagenesis by X-radiation or mitomycin produced a dose-related increase in mutant frequency. HLA-B phenotyping and Southern Analysis of the HLA-A gene suggested that mutation was frequently due to gene deletion, which was often substantial.