The nature of high frequency sister chromatid exchange cells (HFCs)

We employed the three-way differential staining technique (TWD),which allows SCEs to be distinguished on a per generation basisby scoring third metaphases (M3), in order to study the spontaneouslevels of SCEs in normal and high frequency cells (HFCs) thatoccurred in the first (S1), second (S2) and third (S3) S phases.Fifty one of 900 lymphocytes from 37 healthy donors were definedas HFCs by calculating the 95th percentile of the distributionof SCEs in S1 + S2. ‘Normal’ cells presented almostthe same number of SCEs after the first, second and third cellcycles (SCE averages of 2.43, 2.04 and 3.53 respectively). Incontrast, HFCs showed a higher SCE count in SI, which decreasedrapidly through the cycles and reached baseline level at S3(SCE averages of 7.18, 4.29 and 3.45 respectively). This wouldsuggest that the lesions responsible for the higher SCE frequencyin HFCs were effectively removed after two cell cycles and stronglysupport the hypothesis that HFCs are lymphocytes which accumulatehigher levels of DNA lesions through time. ¹To whom correspondence should be addressed     

Sister chromatid exchange distributions in rabbit lymphocytes treated with streptonigrin

Streptonigrin (NSC-45383), a direct-acting clastogen which induces SCEs in vivo and chromosome aberrations both in vivo and in vitro, was evaluated for SCE induction in both G₀ and stimulated rabbit lymphocytes. Determinations were made for 16 cultures from seven female rabbits. These included controls as well as cells exposed to 90 μg/kg in vivo, cells pulse-treated with 50 ng/ml in vitro, and a culture continuously exposed to 5 ng/ml in vitro. For all cultures the SCE/ cell frequency was determined from 20 complete (44 chromosome) metaphases and, in selected cultures, SCEs on individual chromosomes (880 per culture from 20 cells) were enumerated to determine SCE/chromosome frequency and the chromosomal distribution of SCEs. Analysis of variance and least significant difference tests of the √x transformed SCE/cell data show that cells exposed to Streptonigrin while dividing have significantly higher (P<0.01) frequencies (over double the control 5.3 SCE/cell value) whereas treated G₀ cells were not significantly different from the controls. Dispersion analysis of both SCE/cell and SCE/ chromosome data confirms the adequacy of the Poisson distribution for spontaneous or baseline but not streptonigrin-induced SCEs.     

The longevity of chemically induced sister chromatid exchanges in chinese hamster ovary cells

The persistence of the lesions leading to sister chromatid exchanges (SCEs) following acute exposure of Chinese hamster ovary (CHO) cells to direct-acting chemical mutagens was measured. The results revealed that these lesions (and consequently SCEs) are rapidly eliminated from cells. SCE levels fell to near control values by the third or fourth day (six and eight cell cycles, respectively) following exposure of CHO cells to quinacrine mustard (QM) and mitomycin C (MMC). In contrast, cells exposed to methyl methanesulfonate (MMS) showed a small but significant increase in SCE level over control up to and including the fifth day following exposure (approximately ten cell cycles), suggesting that the behavior of these lesions in cells is influenced, at least in part, by the mechanism by which a specific agent interacts with DNA. The possibility that the decline in SCE level was due to the loss of cell populations with high numbers of exchanges was eliminated by the assessment of cloning efficiencies of treated and control cultures.     

Evidence for an indirect effect of radiation on mammalian chromosomes. III. UV- and x-ray-induced sister chromatid exchanges in heterokaryons

The hypothesis that chromosomes may be damaged indirectly by radiation was examined by assaying sister chromatid exchange, (SCE) frequency in heterokaryons between irradiated and unirradiated mouse and Chinese hamster cells. One cell line was UV or x irradiated, then fused to unirradiated BrdU-labeled cells of the other line; SCEs in the unirradiated set were scored in heterokaryons. A dose-dependent increase was consistently observed; the magnitude of which suggested that 25% of UV-induced and up to 60% of x-ray-induced SCEs are indirectly induced. Medium transfer experiments, cell mixing, and fusion with irradiated chick erythrocyte ghosts suggested that unirradiated chromosomes in heterokaryons are damaged by a stable, nondiffusible cytoplasmic component contributed by the irradiated cell.     

Baseline and mitomycin-c-induced sister chromatid exchanges in a melanoma and a colon tumor cell line

Baseline and mitomycin C (MMC)-induced sister chromatid exchanges (SCEs) in two human tumor cell lines (a colon tumor and a melanoma) and in a normal fibroblast cell line were analyzed and compared. The tumor cells showed numerical and structural chromosomal abnormalities. Their baseline SCE rate was slightly, but not significantly, higher than that of the control. Each tumor cell line showed a dose-dependent increase in SCE frequency above the spontaneous level in its own specific manner. The response in the malanoma cell was consistantly below that of the control, but only the response to the highest dose of MMC (10^?9 M) was significantly lower than that of the control. The response of the colon tumor cells varied with respect to that of the control. Thus, it appears that karyotypic instability in tumor cells is not necessarily associated with elevated baseline or induced SCE/chromosome rates. In addition, within each cell line dose group, the SCE frequency was proportional to the number of chromosomes. Thus, the SCE/chromosome is a better expression of genetic damage than SCE/metaphase in analyses involving heteroploid cells.     

Evidence that SCEs induced by mutagens do not occur at the same locus in successive cell cycles: Lack of cancellation in three-way stained CHO chromosomes

An approach based on the synchronization of CHO cells after a first cell cycle incorporating a relatively low amount of bromodeoxyuridine (BrdUrd) into DNA, followed by mutagenic treatment and subsequent culture for second and third generations of BrdUrd incorporation for the scoring of sister chromatid exchanges (SCEs) per cell cycle in three-way differentially (TWD) stained chromosomes, has been used to investigate the possible concellation of SCEs. Cancellation is expected to occur if two mutagen-induced SCEs occur at exactly the same site in subsequent rounds of replication. Lesions in DNA seem to persist and are able to induce SCE throughout two cell cycles after treatment with the three mutagens tested—mitomycin C (MMC), ethyl methanesulfonate (EMS) and ultraviolet (UV) light—though this latter agent was shown as only moderately persistent. Our results seem to indicate that SCEs induced by these mutagens do not take place at the same locus in successive cell generations, as assessed by a lack of SCE cancellation. © 1994 Wiley-Liss, Inc.     

Chromosomal breakage and sister chromatid exchange in peripheral blood lymphocytes and lymphoblastoid cell lines in the X-linked lymphoproliferative syndrome

The frequencies of chromosomal aberrations and sister chromatid exchange (SCE) were measured in lymphoblastoid cell lines (LCLs) and in phytohemagglutinin (PHA)- and pokeweed mitogen (PWM)-stimulated lymphocytes from males with X-linked lymphoproliferative (XLP) syndrome, their obligate carrier mothers, and control subjects. We observed an increased frequency of chromosomal aberrations including increased polyploidy in LCLs derived from families with XLP with time in culture. The SCE rate in LCLs (mean of 3.89 SCEs per cell) was much lower than that in PHA- or PWM-stimulated lymphocytes: PWM-stimulated lymphocytes showed 9.58 SCEs per cell and PHA-stimulated cells had 11.38 SCEs per cell. A greater number of chromosomal gaps and breaks in the D-group chromosomes of LCLs of affected males and carrier females were identified compared to the number expected, based on chromosomal length and the number of aberrations seen in PHA-stimulated cell cultures. No differences in the frequency of SCEs or chromosomal aberrations were found in control subjects and affected males or carrier females in the peripheral lymphocytes stimulated by PHA. Phenotypes of XLP appear to arise from failure of immune responses to Epstein-Barr virus (EBV) and not from intrinsic chromosomal breakage or instability.     

Growth rate and sister chromatid exchange (SCE) incidence of bone marrow cells in Acute Myeloblastic Leukemia (AML)

The sister chromatid exchange (SCE) incidence and growth kinetics have been studied by means of an in vitro bromodeoxyuridine (BrdU) chromosome labeling method in the bone marrow cells of 17 acute myeloblastic leukemia (AML) patients with only diploid cells at diagnosis, remission, and relapse of the disease. At diagnosis, the cells tended to exhibit a low SCE frequency as compared to that during remission. An increased SCE frequency was observed after chemotherapy during remission or relapse. At diagnosis and relapse, when leukemic blast cells predominated in the marrow, they were characterized by the predominance of cells that had undergone only one cell cycle after BrdU exposure. In contrast, the marrow cells during remission tended to resemble the control pattern of growth kinetics, with a predominance of cells undergoing second and third cell cycles in the presence of BrdU. These results suggest that the growth rate of leukemic and nonleukemic cells is different, and that chemotherapy can cause an increased SCE frequency in the marrow cells of AML patients irrespective of the state of the disease.     

Induction and reduction of sister chromatid exchange by CCNU in human lymphocytes in vitro

Sister chromatid exchange (SCE) was studied in human lymphocytes treated with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in vitro. A dose-dependent increase of SCE was observed in cells exposed to 10(-5) - 10(-4) M CCNU. The maximal increase was 25-35 SCEs/cell over the control level, which is similar to the increase found in patients treated with CCNU in vivo. In the presence of rat liver microsomes (S-9 fraction) the frequency of CCNU-induced SCE was slightly higher than in parallel cultures without S-9, suggesting that microsomal metabolism may enhance the rate of decomposition of CCNU into reactive products. The CCNU-induced increase of SCE was greater in cells treated for longer time periods (up to 70 hr) than in cells subjected to a 1-hr treatment. This effect was most pronounced at higher concentrations of the drug (5 X 10(-5) M). The frequency of CCNU-induced SCE was also found to be dependent on the time of treatment in the cell cycle. A treatment for 1 hr during early G1-phase (about 20 hr before the first S-phase) gave rise to a higher increase of SCE than a 1 hr treatment immediately before or during the first or second S-phase. Thus, the CCNU-induced DNA damage leading to SCE seems to persist and may even increase during the prereplicative phase of the cell cycle. After replication in BrdUrd-free medium, the frequency of CCNU-induced SCEs decreased to the control level. The present results, taken together with other studies of strand break and cross-link formation by CCNU in mammalian cells in vitro, suggest that the major SCE-inducing damage by CCNU is DNA interstrand cross-links. These lesions then appear to be slowly removed, if at all, during the prereplicative phase of the cell cycle, and to disappear during or after replication in BrdUrd-free medium in vitro.     

Antipain-mediated suppression of sister chromatid exchanges induced by an inhibitor of poly (ADP-ribose) polymerase

Exposure of mammalian cells to inhibitors of poly(ADP-ribose) polymerase, such as 3-aminobenzamide (3AB) results in the induction of sister chromatid exchanges (SCEs). The mechanism for the induction of SCEs by 3AB is unknown but is thought to be related to the incorporated halogenated pyrimidine used in SCE analysis. In this characteristic, 3AB-mediated SCE induction is similar to the elevated SCE frequency found in Bloom's syndrome (BS) cells. Recently, it has been reported that certain protease inhibitors, such as antipain, will inhibit SCE induction in BS cells. We now report that antipain will also suppress 3AB-induced SCE frequency. As has been reported for BS cells, the effects of antipain on SCE induction are partial, reducing SCE frequency by 0.15 to 0.40 SCE/chromosome (5-25% of the total induced frequency), and 30 microM concentrations of antipain are saturating. Antipain has no effect on baseline SCE frequency. These effects appear to involve free-radical production because dimethylsulfoxide (DMSO), a free-radical scavenger, will mimic the effects of antipain on 3AB-induced SCEs. Both antipain and DMSO will also reduce the elevated SCE frequency found in cells exposed to high (100 microM or more) levels of bromodeoxyuridine (BrdUrd). High exogenous levels of BrdUrd produce some of the same biological effects as 3AB exposure. Thus, a minor fraction of the elevated SCE frequency seen in cells exposed to 3AB or to high levels of BrdUrd appears to be similar to that found in cultured BS cells and is probably due to some free-radical-producing process.     

Establishment of B-lymphoid cell lines retaining cytogenetic characteristics of Bloom syndrome

The present study describes the establishment of and chromosomal changes in B-lymphoid cell lines from cells of Bloom syndrome (BS) patients using Epstein-Barr virus (EBV). Even though PHA-stimulated BS lymphocytes from all five patients studied showed high levels of sister chromatid exchange (SCE), three EBV-transformed BS-B-lymphoid cell lines had normal levels of SCE and two yielded two types of cell populations, i.e., one with increased SCE and chromosome instability (including breaks and quadriradials) and another with normal levels of SCE and without structural aberrations. The karyotypic abnormalities, as observed in the BS lines have not been seen in the cells of any established normal B-lymphoid lines transformed by EBV and strongly suggest that the chromosome abnormalities in the BS--B-cell lines with abnormal karyotypes originated in vivo and not through an in vitro effect of EBV. Furthermore, in the EBV-transformed B-cell lines, we found quadriradial formation between sister chromosomes during endomitoses instead of between homologous chromosomes, strongly suggesting that quadriradial formation may be closely related to SCE. The coexistence in BS subjects of abnormal and normal populations of cells with respect to the number of SCE awaits explanation.     

Genetic effects of 2-aminopurine in mammalian cells

The effects of 2-aminopurine (APur) on mutations, sister-chromatid exchanges (SCEs) and proliferation were investigated in V79 cells by means of cytogenetic and flowcytometric experiments. APur did not induce SCEs after a 3-h treatment before the addition of BrdUrd but SCE frequencies were increased after treatment for two cell cycles in the presence of BrdUrd. SCEs were mainly produced during the second cell cycle of the SCE experiment when BrdUrd substituted DNA is replicated. APur also caused a high percentage of polyploid cells. Compared on the basis of DNA content, SCE induction was the same in diploid and tetraploid metaphases. APur-induced SCEs are strongly influenced by nucleosides. The presence of deoxycytidine (dCyd) caused a reduction of AP-induced SCEs to about control level while addition of deoxythymidine (dThd) enhanced SCE induction. Flow cytometric measurements revealed a small increase in S-Phase cells and a strong accumulation in G2/M after APur treatment in the presence of BrdUrd. S-phase delay was strongly enhanced when BrdUrd substituted DNA is replicated. Addition of dCyd removed the APur-induced inhibition of S-phase in both protocols. Using the same treatment protocol, APur also induced mutations at the HPRT locus. In contrast to their effects on SCEs and proliferation neither BrdUrd nor dCyd had an effect on APur-induced mutations, and dThd reduced the mutation frequency. The results demonstrate that APur-induced SCEs and mutations occur independently from each other. APur-induced mutations obviously occur by a mispairing mechanism while SCEs are a consequence of pool imbalances during replication.     

The effect of the male contraceptive agent Gossypol on human lymphocytes in vitro: traditional chromosome breakage, micronuclei, sister chromatid exchange, and cell kinetics.

The male antifertility agent Gossypol did not affect the level of traditional chromosome breakage or number of micronuclei in 66-hour lymphocyte cultures at concentrations up to 40 micrograms/ml. It did increase the frequency of SCE slightly, although the inter-individual variation was greater than the increase resulting from Gossypol, and, even at the highest concentration (40 micrograms/ml), the SCE rate was still within the normal range. It also affected cell kinetics, reducing the mitotic index and the proportion of second and third metaphases after BUdR incorporation.     

Vitamin E prevents ethylene bis(dithiocarbamate) pesticide zineb-induced sister chromatid exchange in Chinese hamster ovary cells

The in vitro effect of the antioxidant alpha-tocopherol, vitamin E, on deleterious effects induced by the dithiocarbamate fungicide zineb and its commercial formulation azzurro on Chinese hamster ovary (CHO) cells was studied by using frequency of sister chromatid exchanges (SCEs), cell cycle progression and mitotic index (MI) as genetic end points. Both zineb and azzurro activities were tested within the range 0.1-100.0 microg/ml on exponentially growing CHO cells preincubated for 24 h in the presence or absence of 50.0 microg/ml vitamin E. SCE frequencies increased significantly over control values in a concentration-dependent manner in zineb- and azzurro-treated cultures at concentrations of 0.1-10.0 and 0.1-25.0 microg/ml, respectively. When target cells were preincubated with vitamin E, the number of SCEs was significantly lower than that observed in cells exposed only to 1.0-10.0 microg/ml zineb or 1.0-25.0 microg/ml azzurro, but higher than control values. Cytotoxicity was observed at concentrations higher than 25.0 and 50.0 microg/ml zineb and azzurro, respectively, regardless of the absence or presence of vitamin E. Regression analysis showed that the proliferative rate index decreased as a function of the concentration of zineb (0.1-10.0 microg/ml concentration range) and azzurro (0.1-25.0 microg/ml concentration range) titrated into cultures. For both chemicals, progressive concentration-related inhibition of the mitotic activity from cultures was observed when 10.0 microg/ml zineb or 1.0-25.0 microg/ml azzurro was employed. However, no significant alteration in cell cycle progression or MI was observed between vitamin E-preincubated cultures and those treated only with zineb and azzurro.     

Cytogenetic and cytokinetic analysis of lymphocytes from patients with hereditary adenomatosis of the colon and rectum

The frequencies of chromosome aberrations, sister chromatid exchanges (SCEs), and cell cycle kinetics were examined in cultured lymphocytes from five patients with hereditary adenomatosis of the colon and rectum (ACR) [three patients with Gardner's syndrome (GS) and two patients with familial polyposis coli (FPC)]. The frequency of numerical chromosome aberrations was no different in metaphase cells at the first and second replication cycles (M1 and M2) in the ACR patients and control subjects. The percentage of structural chromosome aberrations in both M1 and M2 cells was somewhat higher in the ACR patients as compared with the controls. Neither spontaneous nor mitomycin C-induced SCE frequencies in the patients with ACR were different from the controls, except for one patient with GS, who showed a remarkably high spontaneous SCE frequency. This patient is the mother of a son who had hepatoblastoma. The cell replication index (RI) was lower in the GS patients than in the controls. However, the RI in the FPC patients did not differ from that of the controls.     

Growth, DNA repair, sister chromatid exchange and chromosome studies in fibroblasts fromHuntington's disease patients

Fibroblast cultures from six unrelatedHuntington's Disease (HD) patients and controls and one affected relative of an HD patient were used in studies of cell growth, DNA repair, sister chromatid exchange (SCE) and chromosome aberrations.
There were no significant differences in background levels of SCEs or of chromosome aberrations between HD cultures and controls.
Preliminary results using epidermal growth factor indicated that HD cells may have a lowered relative response to this polypeptide hormone.
Cell growth studies showed no correlation between growth rate and HD.
Increased cell saturation density was recorded in cell lines from four of the HD patients; the remaining three lines from affected individuals (two of them related) were indistinguishable from control cultures. This variation may reflect genetic heterogeneity in HD.
An apparent deficiency in DNA repair capacity following UV irradiation in cultures from three HD patients was subsequently shown to be the result of the increased cell saturation densities in these cultures.     

Enhanced expression of stomach cancer antigen derived from malignantly transformed bloom syndrome cells previously labeled with bromodeoxyuridine.

Bromodeoxyuridine (BrdU) greatly enhanced expression of stomach (ST) cancer antigen (CA) that originated from a malignantly transformed Bloom syndrome (BS) cell line (BS-SHI-4M), although the expression was suppressed with a decrease in sister chromatid exchange (SCE) in the presence of deoxythymidine (dT) or deoxycytidine (dC) and enhanced with an increase in SCE with deoxyguanosine (dG) or deoxyadenosine (dA). Although the exact mechanisms for enhancing CA by BrdU treatment are unknown, these findings appeared to be of special interest because of the parallelism of CA expression and SCE alterations. The finding that BrdU enhancement of the ST CA was effective not only in the immunofluorescence (IF) protocol but also in the band appearance of Western blotting would be worthwhile as a sensitive serodiagnosis of cancer. The 118-kd band obtained from proteins of ST CA cells previously labeled with BrdU was clearly more darkly stained than that from nonlabeled cells and enabled eight weak-positive ST CA to show strong-positive levels retaining complete negativity to nonmalignant sera. Some ST cancer sera (advanced cancer), which originally gave a negative reaction in the nonlabeled condition, still inhibited negative reaction even in BrdU-labeled ST CA cells, however. The inability to detect cancer antibody in our assay might be due to immunocomplexes. Acid dissociation and ultrafiltration of sera from six of seven advanced ST cancers (originally IF negative) have allowed detection of antibody responses to ST CA by Western blot assay with enhanced reactivity as compared with the negativity under native serum conditions. This technique provides a reasonable avenue for study of the mechanisms of CA expression and serodiagnosis.     

Induction of sister chromatid exchanges and inhibition of cellular proliferation in vitro. I. caffeine

Cytogenetic assay systems based on the detection of sister chromatid exchanges (SCE) are widely advocated as a sensitive screening method for assessing genotoxic potential. While many agents have been examined for their ability to induce SCE's, complete dose-response information has often been lacking. We have reexamined the ability of one such compound — caffeine — to induce SCEs and also to inhibit cellular proliferation in human peripheral lymphocytes in vitro. An acute exposure to caffeine prior to the DNA synthetic period did not affect either SCE frequency or the rate of cellular proliferation. Chronic exposure to caffeine throughout the culture period lead to both a dose-dependent increase in SCEs (SCE_d or doubling dose = 2.4 mM; SCE₁₀ or the dose capable of inducing 10 SCE = 1.4 mM) and a dose-dependent inhibition of cellular proliferation (IC₅₀ or the 50% inhibition concentration = 2.6 mM). The relative proportion of first generation metaphase cells, an assessment of proliferative inhibition, increased linearly with increasing caffeine concentrations. However, SCE frequency increased nonlinearly over the same range of caffeine concentrations. Examination of the ratio of nonsymmetrical to symmetrical SCEs in third generation metaphase cells indicated that caffeine induced SCEs in equal frequency in each of three successive generations. The dependency of SCE induction and cellular proliferative inhibition on caffeine's presence during the DNA synthetic period suggests that caffeine may act as an antimetabolite in normal human cells. The significance of these results in regard to both caffeine's genotoxic potential and to the reliability of the SCE assay system are discussed.     

Contribution of O6-alkylguanine and N-alkylpurines to the formation of sister chromatid exchanges,chromosomal aberrations,and gene mutations: New insights gained from studies of genetically engineered mammalian cell lines

O⁶-methyl- and O⁶-ethylguanine are the major premutagenic and precarcinogenic lesions induced in DNA by monofunctional alkyiating agents, albeit formed in minor amounts. The involvement of these lesions in SCE and aberration formation is less clear. We have analyzed the contribution of O⁶-alkylguanine to SCE and aberration formation, as well as its toxic and point mutation inducing effect in transgenic Chinese hamster ovary (CHO) cell lines that express variable amounts of human O⁶-methylguanine-DNA methyltransferase (MGMT). Cells that overexpress MGMT (or the bacterial Ada protein) gained resistance to the formation of alkylation-induced SCEs and aberrations, as compared to MGMT deficient cells. A correlation was apparent between the level of protection for SCEs and cell killing, indicating that both phenomena are interrelated. The protective effects were dependent on the level of MGMT expression, the agent used for alkylation, and cell cycle progression. Our data suggest that at least 2 kinds of lesions are responsible for SCE and aberration formation, namely, O⁶-alkylguanine and one or various N-alkylation products. The probability that O⁶-methylguanine is converted into cytogenetic effects has been estimated to be about 1:30 for SCEs, and 1:147,000 and 1:22,000 for chromosomal aberrations in the first and second post-treatment mitosis, respectively. The induction of SCEs and likely also of aberrations by O⁶-methylguanine requires two replication cycles and is supposed to involve the formation of secondary DNA lesions. Increased repair of 3-methyladenine and 7-methylguanine in CHO cells that overexpress the N-methylpurine-DNA glycosylase (MPG) after transfection with the human MPG-cDNA did not give rise to protection against methylation-induced SCEs and aberrations, probably because of incomplete excision repair. MPG overexpressing cells reacted even more sensitively to methylating agents, suggesting apurinic sites formed as a result of MPG action to be SCE and aberration-inducing lesions. © 1993 Wiley-Liss, Inc.     

Sister chromatid exchange (SCE) rates in human melanoma cells as an index of mutagenesis

Melanomas are highly clonogenic. Genetic variability and polymorphismof tumour cell populations have been reported. However, no directevidence of imitator activity as a source of genetic polymorphismfor melanoma cells has been described. Some intermediates ofmelanin synthesis are cytotoxic and genotoxic and their mutagenicpower has been described. We show here that the rate of sisterchromatid exchange (SCE) of the line of human melanoma cellsused varies with the concentration of the melanin precursorL-tyrosine, in the culture medium. An increase of melanin synthesisresults in increased SCE rates. The highest values of SCEs arefound in melanotic melanoma cells compared with the amelanoticones. Indeed we present evidence that melanoma cells show higherlevels of SCE when compared with normal human lymphocytes, andto the SCE frequencies derived from the literature on the lymphocytesof familial malignant melanoma, sporadic malignant melanomapatients and the lymphocytes of relatives of familial and sporadicmelanoma patients. ³To whom correspondence should be addressed