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.

Mutation rate of normal and malignant human lymphocytes.

The genetic stability of normal and neoplastic lymphocytes was compared by using base-line mutation frequency and mutation rate/cell generation. Mutations at the hypoxanthine-guanine phosphoribosyltransferase locus were studied by enumerating thioguanine-resistant cells in a clonogenic assay. The base-line ("spontaneous") mutation frequency was 1.52 X 10(-6), 6.38 X 10(-6), and 1.06 X 10(-6) for normal cells from three individuals and was 1.16 X 10(-3), 6.08 X 10(-5), and 3.06 X 10(-5) for the three malignant cell lines, Jurkat (JM), HRIK, FMC-Hu1B, respectively. The mutation cell/generation rate was 24.6 X 10(-8), 15 X 10(-8), and 5.5 X 10(-8) for lymphocytes from the three normal individuals, and 666.4 X 10(-8), 52.8 X 10(-8), and 131 X 10(-8) for the three malignant cell lines. The results suggest that neoplastic lymphocytes are more genetically unstable than normal lymphocytes.

In vivo somatic mutations in human lymphocytes frequently result from major gene alterations.

Somatic mutations, either spontaneous or produced by identifiable mutagens, are thought to be important in the aetiology of cancer and in the ageing process. The study of somatic mutations in human cells in vivo has recently been made possible by the development of techniques for enumeration and clonal expansion of lymphocytes mutated at the chromosome X-linked hypoxanthine phosphoribosyl transferase (HPRT) locus. We have studied the molecular basis of in vivo hprt mutations in human lymphocytes and report here that a surprisingly high proportion (57%) involve substantial gene alterations which are not evident cytogenetically. These major gene alterations include deletions, exon amplifications and novel, sometimes amplified, bands on Southern analysis. Such changes emphasize the fluid nature of information in DNA and may be indicative of general mechanisms by which functional gene loss is involved in the aetiology of cancer and the homeostatic failure of ageing.

Sensitivity of lymphocytes from patients with cancer to x- and ultraviolet radiation.

It has been suggested that radiation sensitivity may be increased in certain patients with cancer. Using a high-efficiency limiting dilution cloning technique, the sensitivity to x- and ultraviolet (UV)-radiation of lymphocytes from untreated patients with cancer to that of age-matched controls was compared. Sensitivity to x-radiation in 19 patients with carcinoma and 10 with lymphoma, and sensitivity to UV-radiation in 8 patients with carcinoma and 4 with lymphoma were studied. The proportion of unirradiated lymphocytes forming clones was significantly lower in patients with cancer, particularly lymphoma, but there was no suggestion of increased sensitivity to either x- or UV-radiation in any of the patients studied.

The cells of origin of lymphocyte clones grown by limiting dilution.

A high proportion of the mononuclear cells separated from peripheral blood by Ficoll-Hypaque centrifugation show extensive proliferation when cloned at limiting dilution. The nature of clone-forming cells ( CFCs ) and the cells generated during clone formation were studied by cytotoxicity with antibody and complement and by immunofluorescence. Cytotoxicity prior to cloning with OKT3 plus OKT11 eliminated 99.34% of clones, indicating that virtually all CFCs are T-lymphocytes, and cytotoxicity with OKT4 or OKT8 indicated that helper and suppressor subclasses each contribute approximately half of the CFCs . Immunofluorescence of cells proliferating in clones confirmed that all clones were T-lymphocytes and showed that 47% were OKT4 positive and 47% were OKT8 positive; 6% were negative with both OKT4 and OKT8. The results indicate that in the nearly optimal conditions for proliferation provided by limiting dilution, clones arise entirely from T-lymphocytes and clone formation is a property of a variety of lymphocyte subclasses.

SCE, X-radiation sensitivity and mutation rate in multiple sclerosis.

In order to detect any underlying DNA abnormality that may be present in multiple sclerosis (MS), the incidence of SCE, X-radiation sensitivity and the frequency of 6-thioguanine-resistant cells were determined in the lymphocytes of 34 MS patients. As a group, MS patients showed an increase in the SCE rate compared to control. However, there was no increase in X-radiation sensitivity. The frequency of 6-thioguanine-resistant mutant cells was also normal.

Effect of age on sensitivity of human lymphocytes to radiation. A brief note.

Survival of human lymphocytes after X-ray and ultraviolet irradiation was studied in individuals of various ages using a technique of cloning in soft agar. There was no effect of age on survival after ultraviolet irradiation but lymphocytes from elderly individuals were approximately twice as sensitive to X-radiation as lymphocytes from young individuals. The results are relevant to the possible importance of DNA damage and repair in cellular ageing.

Radiation sensitivity of human lymphocytes.

A simple and rapid in vitro technique to assess the sensitivity of human lymphocytes to X- and UV-radiation is described. Peripheral blood lymphocytes are cloned in agar and colony survival is used as the end point. Survival down to a fraction of approx. 0.02 can be measured accurately. The D10 for X-radiation is approx. 165 rad and the initial portion of the survival curve is linear with no evidence of a threshold. The D10 for UV-radiation is approx. 50 erg/mm2. The technique should be of value both for general radiobiological studies and for detecting individuals having abnormalities of DNA repair.