Complementation analysis in ataxia telangiectasia: fibroblast-lymphoblastoid cell heterokaryon assay by radiation-induced chromosome aberrations.

Using radiation-induced chromosome aberrations as a marker, heterokaryons of ataxia telangiectasia formed between appropriate combinations of lymphoblastoid cell lines (LCLs) and fibroblasts representing four and two different groups, respectively, were analyzed. The results showed that complementation group 2 in the LCL series corresponds to group D in the fibroblast series.

Detection of somatic mutations in tumours of diverse types by DNA fingerprinting with M13 phage DNA.

Hybridization of M13 phage DNA to Southern blots of human DNA produces an individual-specific DNA fingerprint. In this study, tumour and lymphocyte DNA from a series of patients with melanoma, Merkel-cell carcinoma, Burkitt's lymphoma and Wilms' tumour was probed with M13 DNA to detect somatic mutations in the DNA of the tumours. Somatic changes were observed in tumour DNA of 16 out of the 28 cases examined. This frequency compared favourably with the frequency with which tumour-specific changes have been found when using the Jeffreys DNA fingerprinting probe 33.15, and demonstrates that M13 DNA provides a useful additional probe for the study of somatic changes in tumours. The finding that multiple DNA fragments were lost or gained in DNA fingerprints from individual tumours indicates a marked degree of complexity in the genetic changes involved in the evolution of certain human cancers.

The EcoRI RFLP of c-mos in patients with non-Hodgkin's lymphoma and acute lymphoblastic leukemia, compared to geriatric and non-geriatric controls.

We have used Southern blot analysis to type individuals for the presence or absence of a rare EcoRI RFLP at the c-mos proto-oncogene locus. This polymorphism has previously been reported to be associated with cancer. Ninety-eight patients with non-Hodgkin's lymphoma (NHL) or acute lymphoblastic leukemia (ALL) and 154 cancer-free individuals, including 108 geriatric patients with no family history of cancer, were studied. Because 4 geriatric patients (aged 67-94) were found to have the rate c-mos allele (A2), and the frequency of this A2 allele was no higher among the lymphoma/leukemia patients than among cancer-free individuals, it is unlikely that it constitutes a marker for NHL or ALL.

Allelic variation of the c-raf-1 proto-oncogene in human lymphoma and leukemia.

Several lines of evidence point to the involvement of the proto-oncogene c-raf-1 in the development of lymphoma and leukemia and in a previous study we found variants of this gene in 3 lymphoma patients. To see whether variation at this locus plays a role in predisposition to these cancers, we have examined the frequency of an unusual EcoRI allele of c-raf-1 in 99 non-Hodgkin's lymphoma and 28 acute lymphoblastic leukemia patients, and in 182 controls. To optimize the chance of detecting a difference, we selected 113 of the controls from geriatric patients with no family history of cancer. No difference in the frequency of the allele was found between patients and controls, thereby refuting our hypothesis that this polymorphism of the c-raf-1 locus contributes to genetic susceptibility to these two related cancers. We estimate that the frequency of the rarer b EcoRI allele of this locus is 0.091 +/- 0.012 in the Australian Caucasian population.

A Taqi RFLP of the human TGF alpha gene is significantly associated with cutaneous malignant melanoma.

A TaqI restriction fragment length polymorphism (RFLP) of the human transforming growth factor alpha (hTGF alpha) locus was analyzed in DNA from 63 normal individuals, 34 malignant melanoma (MM) cell lines, and 18 melanoma biopsy specimens. The frequency of a 2.7-kb allele (0.18) in MM cell lines was significantly higher (p less than 0.01) than in lymphoblastoid cell lines (LCLs) derived from unaffected controls (0.05). The frequency (0.14) in MM biopsies was similar to that in MM cell lines although, owing to the small numbers investigated, it was not significantly higher than in controls. In the case of 5 MM patients who were constitutionally heterozygous for alleles at the TGF alpha locus, no apparent losses of heterozygosity were observed in the corresponding tumour DNA. Thus, the constitutional presence of the 2.7-kb allele may be a risk factor for melanoma.

c-Ha-ras-1 alleles in bladder cancer, Wilms' tumour and malignant melanoma.

Polymorphism of the human c-Ha-ras-1 gene has been analysed in DNA from 168 individuals using the enzymes MspI and HpaII. In all, 35 bladder cancer patients, 28 melanoma patients, 22 Wilms' tumour patients, 24 first-degree relatives of Wilms' tumour or melanoma patients and 59 unaffected controls were studied. A total of 13 different fragment sizes was detected, 4 "common" and 9 "unusual". Of the latter, 4 were observed only in cancer patients or their first-degree relatives. The frequency of unusual alleles was significantly greater in bladder cancer patients and in the combined tumour group than in controls, thus providing support for the association of unique Ha-ras alleles and cancer. Some unaffected relatives of patients carried unusual alleles, and thus there is no absolute relationship between Ha-ras genotype and disease.

Sensitivity to ultraviolet radiation in a dominantly inherited form of xeroderma pigmentosum.

An Australian family is described in which a mild form of xeroderma pigmentosum (XP) is inherited as an autosomal dominant trait. Studies of lymphoblastoid cells and fibroblasts from affected persons demonstrated cellular sensitivity to ultraviolet (UV) light as judged by diminished clonogenicity and higher frequencies of UV induced chromosome aberrations compared to normal controls. After UV irradiation of dominant XP cells, replicative DNA synthesis was depressed to a greater extent than normal and the level of UV induced DNA repair synthesis was lower than that in normal cells. The level of sister chromatid exchanges and the numbers of 6-thioguanine resistant mutants induced by UV irradiation were equal to those found in normal controls. Although two subjects in the family had skin cancers, this dominant form of XP is not apparently associated with high risk, or large numbers, of skin cancers in affected persons.

Wilms' tumour: association with cellular sensitivity to mitomycin C in patients and first-degree relatives.

To investigate whether predisposition to Wilms' tumour is associated with a particular defect in the handling of DNA damage, cell-lines from families in which the tumour had occurred were tested for sensitivity to a variety of DNA-damaging agents. Lymphoblastoid lines from both Wilms' tumour patients and their first-degree relatives showed increased sensitivity to the cross-linking agent, mitomycin C, but normal sensitivity to ultraviolet (UV) and gamma irradiation. Thus sensitivity to mitomycin C damage can be associated with the Wilms'-tumour-susceptible genotype and could be a genetic factor responsible for the modification of expression of this genotype.

Effects of ultraviolet irradiation on the cell cycle in normal and UV-sensitive cell lines with reference to the nature of the defect in xeroderma pigmentosum variant.

Analysis of the distribution of cells through the phases of the cell cycle by DNA flow cytofluorimetry has been utilized to investigate the effects of ultraviolet (UV) irradiation on cell-cycle progression in normal and UV-sensitive lymphoblastoid cell lines. In time-course studies only slight perturbation of DNA distribution was seen in normal cells, or UV-sensitive familial melanoma (FM) lines in the 48 h following irradiation. Xeroderma pigmentosum (XPA) excision-deficient cells showed a large increase in the proportion of cells in S phase 16-40 h post-irradiation. XP variant (XPV) cells were blocked in G1 and S phases with the complete absence of cells with G2 DNA content 16-28 h after irradiation. By 48 h post-irradiation the DNA distribution of XPA and XPV cells had returned to that of an unirradiated control. When colcemid was added to the cultures immediately after irradiation to prevent mitotic cells dividing and re-entering the cell cycle, progression through the first cycle after irradiation was followed. UV irradiation did not affect the rate of movement of cells out of G1 into S phase in normal, FM or XPA cells. The proportion of cells in S phase was increased in UV-irradiated cultures in these cell types and the number of cells entering the G2 + M compartment was reduced. In UV-irradiated cultures of XPV cells a large proportion of cells was blocked in G1. The rate of accumulation of cells with G2 DNA content was equal to that of the control until 4 h post-irradiation, thereafter falling below the control. Thus XPV cells in S phase at the time of irradiation complete DNA synthesis to reach G2 DNA content. However, cells irradiated in G1 are blocked from entry into S. These results suggest that there is a defect in XPV cells that affects a step prior to the onset of DNA replication.

Familial melanoma associated with dominant ultraviolet radiation sensitivity.

Sensitivity to ultraviolet radiation was studied in lymphoblastoid cell lines derived from 32 members of two families with histories of multiple primary melanomas in several generations. As assayed by colony formation in agar or by trypan blue exclusion following irradiation, cellular sensitivity showed a bimodal distribution. All persons with melanoma or multiple moles were in the sensitive group, while some family members exhibited responses similar to those of controls. Cells from four cases of sporadic melanoma showed normal levels of sensitivity. The data are consistent with a dominantly inherited ultraviolet light sensitivity associated with these examples of familial melanoma. Spontaneous and ultraviolet light-induced sister chromatid exchange frequencies were similar to those in control cell lines. No defect in excision repair was detected in any of the above cell lines, but the sensitive group showed postirradiation inhibition of DNA replication intermediate between controls and an excision-deficient xeroderma pigmentosum cell line.

Ultraviolet radiation sensitivity of proliferating and differentiated human neuroblastoma cells.

The effects of ultraviolet (U.V.) radiation were studied on a cloned line of human neuroblastoma cells in proliferative and differentiated growth modes, the latter being induced by serum deprivation. The neuroblastoma cells were found to be unusually sensitive in comparison with HeLa cells when survival was measured by colony formation in soft agar, the differentiated mode being the most sensitive. Ultraviolet radiation sensitivity was associated with very low DNA repair capacity as measured by DNA repair synthesis and by removal of M. luteus endonuclease-sensitive sites from irradiated DNA. The greater sensitivity of the differentiated cells appeared to be related to a greater degree of DNA damage at a given U.V. dose, resulting from altered cell geometry in the growth mode. The neuroblastoma cells showed little or no post-irradiation inhibition of DNA replication at low U.V. doses, suggesting that it is the repair process rather than the DNA damage which is responsible for inhibiting replication.

Sensitivity and resistance of human melanoma cells to ultraviolet radiation.

A human melanoma cell line (MM253) was found to be sensitive to ultraviolet (UV) radiation, having a Do of 1.0 J/M2 when cloned on plastic culture dishes and a Do of 2.3 J/m2 when cloned in agar. These figures are much lower than those obtained for all other human melanoma cell lines studied in this laboratory (Do of 32-40 J/m2) and demonstrate that MM253 is unusually UV sensitive. The increased level of UV sensitivity in MM253 is not due to a reduced capacity for excision of pyrimidine dimers, repair of DNA single-strand breaks or elongation of newly-synthesized DNA strands when comparison is made with a UV-resistant melanoma cell line.

Mammalian cell function mediating recombination of genetic elements.

Recombination of segments of the SV40 genome by a variety of mechanisms is described. These include the faithful joining of linear segments that have flush termini as opposed to previously described cohesive or resected termini. Lack of involvement of viral proteins has been demonstrated for recombination of segments with homologous overlapping termini, but probably applies also to the other joining reactions. Segments of the genome that have been cleaved in such a manner as to be unable to manufacture any known viral proteins are neutral elements of genetic information, incapable of selection by replication or biological function until recombined. These recombination functions presumably are available to the host cell and any element of genetic information that can be generated in that cell.