Mutagenesis, Vol. 16, No. 4, 317-322,
July 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Normal human lymphocytes exhibit a wide range of methionine-dependency which is related to altered cell division but not micronucleus frequency
1 Department of Physiology, Adelaide University, SA 5005 and 2 CSIRO Health Sciences and Nutrition, PO Box 10041, Adelaide BC, SA 5000, Australia
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Abstract |
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The underlying cause(s) of methionine-dependency and its relevance to cancer remains unclear. We aimed to determine whether (i) normal human lymphocytes exhibit methionine-dependency, (ii) baseline levels of genetic damage are related to methionine-dependency and (iii) methionine-dependence can be explained, in part, by common polymorphisms in methionine synthase and methylenetetrahydrofolate reductase (MTHFR). Genetic damage was measured in lymphocytes of 52 volunteers (29–65 years) using the cytokinesis-block micronucleus assay. Methionine-dependency was assessed by culturing cells in serum-free media containing 0.1 mM L-methionine and 0 mM D,L-homocysteine (met+hcy–) or 0 mM L-methionine and 0.2 or 0.4 mM D,L-homocysteine (0.2/0.4-hcy+)(met–hcy+). Mitogenesis was stimulated with phytohaemagglutinin. Cytokinesis was inhibited by adding cytochalasin B at 44 h. Ninety-six hours after PHA, cells were transferred to microscope slides. Cell proliferation was measured by counting binucleated cell frequency and calculating nuclear division index. Volunteers were classified into tertiles of methionine-dependence according to the growth of their cells in met–hcy+ media (relative to growth in met+hcy– media). Average cell division, as a percentage of division in met+hcy– media, was approximately 5, 26 and 70% in 0.2-hcy+ media and 29, 70 and 142% in 0.4-hcy+ media for the high, mid and low tertiles of methionine-dependence, respectively. Micronucleus frequency did not vary between these tertiles (P > 0.6). In both met+hcy– and met–hcy+ media, cell division was not affected by polymorphisms in MTHFR (C677T, A1298C) or methionine synthase (A2756G). Cell division in met–hcy+ media was negatively correlated with division in met+hcy– media (P = 0.05 and 0.007 for 0.2 and 0.4-hcy+, respectively). Methionine-dependent lymphocytes had higher levels of cell proliferation in met+hcy– media than methionine-independent lymphocytes (P = 0.089 and 0.01 for 0.2 and 0.4-hcy+, respectively). However, this difference was not apparent in previous experiments when cells were grown in media containing 10% fetal calf serum. These findings show that there is a wide inter-individual variation in the degree of methionine-dependency of normal human lymphocytes in vitro. Methionine-dependency does not appear to alter the risk for chromosomal mutation as measured by the micronucleus assay. We discuss the possible relevance to cancer of increased cell division in methionine-dependent cells under methionine-replete and serum-free media conditions.
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Introduction |
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Methionine-dependence is the inability of cells to proliferate when methionine is replaced by homocysteine, one of its immediate precursors. This phenotype has been reported in numerous cancer cell lines (Hoffman, 1982
; Stern et al., 1984) and human tumors (Hoshiya et al., 1995) and has long been considered a property unique to cancer cells (Hoffman, 1985). For this reason, treatments involving methionine-restriction have been proposed as a potential method for controlling the growth of methionine-dependent cancer cells (Breillout et al., 1990; Guo et al., 1993; Hoshiya et al., 1995).
It is becoming evident that methionine-dependency is not solely a property of cancer cells (Kano et al., 1982; Mikol and Lipkin, 1984; Christa et al., 1986; Judde and Frost, 1988). For example, Mikol and Lipkin (1984) report that non-cancerous skin fibroblasts taken from patients with hereditary colon cancer are relatively methionine-dependent compared with those of controls with no familial history of colon cancer. Because methionine-dependency can exist in both normal and cancer cells in the same individual it seems possible that methionine-dependent cancer cells simply develop in individuals who were originally methionine-dependent. In this scenario, methionine-dependency is a pre-existing phenotype that is not acquired during transformation. Whether methionine-dependence is a risk factor for cancer is not yet clear.
The culturing of methionine-dependent cells in methionine deficient, homocysteine supplemented (met–hcy+) media results in a reversible late cell cycle arrest (Hoffman and Jacobsen, 1980; Guo et al., 1993). Elevated levels of transmethylation (Figure 1), and hence a higher demand for methyl groups, is one of the proposed mechanisms for methionine-dependent cell cycle progression (Hoffman, 1985; Judde et al., 1989).
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Methionine-dependent cells are reported to have lower S-adenosyl methionine (SAM) levels than methionine-independent cells (Stern et al., 1984
), a finding consistent with elevated rates of transmethylation. Because SAM is an inhibitor of methylenetetrahydrofolate reductase (MTHFR) (Kutzbach and Stokstad, 1971), a decline in SAM levels may accelerate the conversion of 5,10-methylene tetrahydrofolate (5,10-meTHF) to 5-methyl tetrahydrofolate (5-meTHF) by MTHFR and divert folate away from thymidine synthesis. These conditions may elevate intracellular dUTP/dTTP ratios and subsequently increase the rate of uracil misincorporation into DNA (for review see Blount et al., 1997). The simultaneous removal of adjacent uracil residues, within 12 base pairs of each other, during excision repair may result in the formation of double-stranded DNA breaks (Dianov et al., 1991). The introduction of double-stranded breaks leads to chromosome breakage and rearrangement, events that significantly correlate with the risk for cancer (Hagmar et al., 1994; Hagmar et al., 1998; Bonassi et al., 2000).
The recycling of homocysteine to methionine is catalysed by vitamin B12-dependent methionine synthase in a reaction where 5-meTHF is the methyl group donor (Figure 1
). Theoretically, the reduced activity of methionine synthesis is an attractive mechanism for the aetiology of methionine-dependence. This mechanism is supported by the finding that a methionine-independent revertant cell line had a 31% greater methionine synthase activity than the methionine-dependent cell line from which it was isolated (Judde et al., 1989). Furthermore, of the other cancer cell lines tested methionine synthase activity was at least 20% greater in methionine-independent cells than in methionine-dependent cells (Judde et al., 1989). It is also reported that methionine-dependent glioma cells (P60) remethylate homocysteine to methionine substantially slower than methionine-independent glioma cells (P60H), possibly due to a reduced capacity of P60 cells to furnish methylated cobalamin to methionine synthase (Fiskerstrand et al., 1997).
Occupational exposure to nitrous oxide, an inhibitor of methionine synthase, is reported to cause a significant elevation in micronucleus frequency (Chang et al., 1996). Methionine synthase inhibition causes folate trapping (Horne and Holloway, 1997) and a reduction in the amount of folate available for thymidine synthesis as described above. Furthermore, low vitamin B12 status, the essential cofactor for methionine synthase, is associated with an elevated micronucleus index in vivo (Fenech et al., 1997, 1998). It follows that if reduced methionine synthase activity is a cause of methionine dependency, a cell's methionine requirements may be related to markers of genetic damage (e.g. micronucleus frequency).
Although it is not known whether the common A2756G mutation (Leclerc et al., 1996) in the gene encoding methionine synthase causes a reduction in enzyme activity, it is possible that such a mutation may affect the (exogenous) methionine requirements of normal human lymphocytes in culture. In contrast to the above results which support a role of methionine synthase in methionine-dependency, some earlier studies show that methionine-dependent cells have similar methionine synthase activities to methionine-independent cells (Hoffman and Erbe, 1976; Hoffman et al., 1978; Stern et al., 1984; Christa et al., 1986).
It is also conceivable that mutations in the gene encoding MTHFR could affect a cell's methionine requirements. MTHFR catalyses the conversion of 5,10-meTHF to 5-meTHF, the methyl donor required for the conversion of homocysteine to methionine (Figure 1). Two common mutations in the MTHFR gene, C677T (Frosst et al., 1995) and A1298C (van der Put et al., 1998), are reported to reduce MTHFR activity by 70 and 39%, respectively, in homozygous mutants. This may affect a cell's requirement for exogenous methionine by limiting the remethylation of endogenous homocysteine.
There is a clear need to clarify the cause and effects of methionine dependence, not only in cancer cells but in normal human cells. Indeed the mechanisms behind this phenotype may even vary between cell types. In this study we aimed to determine whether methionine-dependence is related to baseline levels of genetic damage, as measured by the CBMN assay, in normal human lymphocytes. Furthermore, we aimed to determine whether mutations in two key folate-metabolizing enzymes (MTHFR and methionine synthase) affect lymphocyte methionine requirements in vitro.
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Materials and methods |
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Fifty-two healthy asymptomatic people (23 male, 29 female) aged 29–65 years and not receiving anti-folate therapy or cancer treatment were recruited for the study. Each gave their informed consent and ethics approval was obtained from the CSIRO Health Sciences and Nutrition and Adelaide University human ethics committees.
On the first visit, volunteers donated a fasted blood sample (18 ml in lithium-heparinized vacuettes). Lymphocytes were isolated using Ficoll-Paque gradients (Pharmacia Biotech, Uppsala, Sweden) and DNA was isolated by cell lysis followed by proteinase K (Sigma, St Louis, MO, USA) treatment, salt extraction and ethanol precipitation. Baseline levels of genetic damage in lymphocytes were measured using the cytokinesis-block micronucleus assay as described by Fenech (1993). Micronuclei are small nuclear bodies that contain chromosome fragments or whole chromosomes that have not been packaged into the newly formed nuclei during cell division. Micronuclei are observed in binucleated cells in which cytokinesis has been inhibited. Briefly, duplicate lymphocyte cultures were prepared at a concentration of 1x106 cells/ml in 750 µl RPMI 1640 with 10% fetal bovine serum (FBS; Trace Biosciences, Victoria, Australia) for each person. Mitogenesis was stimulated by the addition of phytohaemagglutinin (22 µg/ml) (PHA; Murex Biotech, Kent, UK) and cultures were incubated at 37°C in a humidified incubator with 5% CO2. Forty-four hours later, cytokinesis was inhibited with cytochalasin-B (4.5 µg/ml) (Cyto-B; Sigma). After 72 h, cells were transferred to microscope slides (two spots per slide) by cyto-centrifugation (Shandon Southern Products, Cheshire, UK), air dried and stained using Diff-Quik (gives similar results to Wright-Giemsa stain; Lab Aids, NSW, Australia). Cells were scored on coded slides using the standard criteria (Fenech, 1993) with two scorers, each counting one (of two) spot per slide. The micronucleus frequency of female volunteers was gender adjusted by dividing by a factor of 1.87 (the ratio of the average female micronucleus frequency to male micronucleus frequency in this study). The data were then age adjusted to an age of 49.5 years (average age of volunteers) using the equation proposed by Fenech et al. (1994).
The presence of the MTHFR C677T and A1298C and methionine synthase A2756G mutations was determined using the restriction fragment length polymorphism techniques described by Frosst et al. (1995), van der Put et al. (1998) and Leclerc et al. (1996), respectively. PCR primers were obtained from GeneWorks (Adelaide, Australia) while dNTPs and Taq DNA polymerase were purchased from Roche Diagnostics (Basel, Switzerland).
Plasma homocysteine concentrations in these samples were measured by HLPC with fluorescent detection using the internal standard method of Vester and Rasmussen (1991).
On the second visit, volunteers donated another fasted blood sample (9 ml, in lithium-heparinized vacuettes) and lymphocytes were isolated. Methionine-dependency was assessed by preparing three lymphocyte cultures per person. Each culture contained 1x106 cells in 900 µl RPMI 1640 medium (Trace Biosciences) without FBS or methionine. Cultures were then spiked with 100 µl RPMI 1640 medium containing either 1 mM L-methionine, 2 mM D,L-homocysteine or 4 mM D,L-homocysteine (all from Sigma) in order to achieve concentrations of 0.1 mM methionine (met+hcy–), 0.2 mM homocysteine (0.2-hcy+) and 0.4 mM homocysteine (0.4-hcy+), respectively. PHA (22 µg/ml) was added and cultures were incubated at 37°C. After 24, 44 and 72 h, 100 µl media was removed (and discarded) from cultures and was replaced with 100 µl spiking solution. Cyto-B (4.5 µg/ml) was added after spiking at 44 h. Ninety-six hours post-PHA, cells were harvested onto microscope slides as described above. Cultures were repeatedly spiked (daily) because a single addition of homocysteine does not support cell division (data not shown). Because FBS was not used, cell culture time was increased from 72 to 96 h in order to increase the yield of binucleated cells. A minimum total of 500 cells was scored per slide. Cell division in met+hcy– medium is represented as a percentage of viable cells that are binucleated. Tri- and tetranucleated cells, although infrequent, were regarded as 1.5 and 2 binucleated cells, respectively. For each individual, cell division in met–hcy+ medium is expressed as a percentage of cell division in met+hcy– medium. Nuclear division index was calculated according to the equation of Eastmond and Tucker (1989). The experimental design is illustrated in Figure 2.
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Normality tests were performed in order to determine whether parametric or non-parametric analyses should be employed. Relationships between data were investigated using Pearson correlation tests and multiple regression analyses. Comparisons between groups were made using a one-way ANOVA (with Tukeys' post-test). Analyses were performed using Prism® version 2.01 and InStat® version 3.0, both from GraphPad Software, San Diego, CA, USA. Significance was accepted at P < 0.05.
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Results |
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Data for cell division in met+hcy– and met–hcy+ media exhibited normal distributions. Volunteer age was negatively correlated with division in met+hcy– media (P < 0.001, r2 = 0.19). There was a positive correlation between cell division in 0.2-hcy+ media and 0.4-hcy+ media (P < 0.0001, r2 = 0.46).
Volunteers were sorted into tertiles of methionine- dependence according to the division rate of their cells in met–hcy+ media (relative to division in met+hcy– media). Those in the lowest tertile of growth in met–hcy+ media were classified in the highest tertile of methionine-dependence and vice versa. Two methods of sorting were used, one based on cell division rate in 0.2-hcy+ media and the other on cell division rate in 0.4-hcy+ media. There is no age difference between tertiles for both 0.2-hcy+ and 0.4-hcy+ (Table II).
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Cell division rate in met–hcy+ media was negatively correlated with %BNs in met+hcy– media (P = 0.05, r2 =0.07 and P = 0.007, r2 = 0.14 for 0.2-hcy+ and 0.4-hcy+, respectively). Lymphocytes with the highest methionine-dependence were found to have higher rates of cell division in met+hcy– media than lymphocytes in the lowest tertile of methionine-dependence (Table I
, Figure 3). In other words, increased methionine-dependence was associated with increased cell division in methionine-supplemented, serum-free media compared with methionine-independent lymphocytes. However, the methionine-dependence phenotype was not associated with increased cell proliferation when cells were grown in medium supplemented with 10% FBS (Table III).
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There was no association between age/gender-adjusted micronucleus frequency (Table II
) or plasma homocysteine concentrations (Table III) and methionine dependency phenotype.
The growth of lymphocytes in met+hcy– and met–hcy+ media was not significantly different between homozygous wild-types, heterozygotes or homozygous mutants for all three polymorphisms studied (Table IV). Furthermore, when the values 0, 1 and 2 were assigned to represent the number of mutant alleles it was found that the frequency of mutant alleles was similar between the tertiles of division in met+hcy– and met–hcy+ media (P > 0.05).
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For the MTHFR 677 and 1298 and methionine synthase 2756 loci the frequency of mutant alleles was 38.5% (T), 33.65% (C) and 13.7% (G), respectively. The population studied was found to be in Hardy–Weinberg equilibrium with respect to all three genotypes. The association between these mutations and baseline micronucleus frequency is currently being evaluated on a larger population and will be reported elsewhere.
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Discussion |
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The results of this experiment clearly suggest that methionine-dependence is a common phenotype in lymphocytes taken from normal healthy human beings. At least one-third of the volunteers tested had cell growth rates in met–hcy+ media that were half the rate of cell division in met+hcy– media (Table I
). Mikol and Lipkin (1984) have previously reported that non-cancerous skin fibroblasts taken from patients with hereditary colon cancer are relatively methionine-dependent compared with those of controls with no familial history of colon cancer. However, to the best of our knowledge this is the first report of primary lymphocytes, taken from volunteers with no history of cancer, exhibiting varied degrees of methionine-dependency. Kano et al. (1982) have previously reported that peripheral blood lymphocytes taken from normal volunteers fail to proliferate in met–hcy+ media containing 30 mg/l (220 µM) homocysteine. Experiments in this laboratory have shown that when homocysteine is added to PHA-stimulated lymphocytes in cell culture at a concentration of 200 µM, the amount of homocysteine remaining after 24 h is only 12.77 ± 3.39 µM (n = 8, unpublished data). Furthermore, in other experiments we have observed that a single addition of homocysteine at 200 and 400 µM cannot support proliferation in lymphocytes; however, they grow normally when the homocysteine is added to the cell culture every 24 h (unpublished results). In light of these results, we suggest that the observations made by Kano et al. (1982) cannot be interpreted as methionine-dependency of lymphocytes because it is likely that the amount of homocysteine present in their system could not support the growth of methionine-independent cells, nor methionine-dependent cells.
The most reasonable explanation for methionine-dependency is a failure to convert homocysteine to methionine by methionine synthase, a model supported by some experimental evidence (Judde et al., 1989; Fiskerstrand et al., 1997). We hypothesized that those with a methionine-dependent phenotype may have a higher micronucleus frequency than those with a methionine-independent phenotype, because they may be expected to have a lower intracellular SAM concentration, which would facilitate folate trapping and increase uracil misincorporation into DNA. This hypothesis is not supported by the results of this experiment which show that there is no difference in the age and gender-adjusted micronucleus frequency of people in the highest (methionine-independent) and lowest (methionine-dependent) tertiles of cell division in met–hcy+ media. If methionine synthase deficiency explains the methionine-dependency phenotype, then a possible explanation for the null effect of methionine-dependence on MNi frequency is that methionine synthase deficiency (or inhibition) may result in a build up of homocysteine, S-adenosyl homocysteine (SAH) and S-adenosyl methionine (SAM) when methionine is supplied in the culture medium or diet (Figure 1). The known inhibitory effect of SAM on MTHFR (Kutzbach and Stokstad, 1971) may serve to protect against DNA damage by allowing sufficient availability of 5,10-MeTHF for the efficient conversion of dUMP to dTMP. Therefore, methionine-dependence would only favour a low SAM concentration, elevated dUMP and MNi from chromosome breaks during conditions of methionine deficiency which would also coincide with induction of MNi from chromosome loss events caused by hypomethylation of heterochromatin. It is also possible that differences in micronucleus frequency between methionine-dependence phenotypes may only be detectable under conditions of folate deficiency, because a high folate concentration may facilitate the provision of sufficient 5,10-meTHF for thymidine synthesis despite a methionine synthase deficiency-induced folate trapping. Future studies should be directed towards comparing the genetic stability of methionine-dependent and methionine-independent cells under conditions of methionine and/or folate deficiency conditions that allow cell division to proceed.
The theory that methionine-dependency may be influenced by mutations in key folate-metabolizing enzymes was not supported by the results presented here (Table IV). It is not known to what extent the A2756G mutation affects methionine synthase activity; however, there is evidence indicating that people without the mutation are at a modestly higher risk for hyperhomocysteinemia than hetero- or homozygous mutants (Harmon et al., 1999; Tsai et al., 2000). It is evident that the A2756G mutation (or lack of it) in methionine synthase cannot account for the methionine-dependent phenotype. Furthermore, because the C677T and A1298C MTHFR polymorphisms are known to reduce enzyme activity by 70 (Frosst et al., 1995) and 39% (van der Put et al., 1998) in homozygous mutants, respectively, we suggest that MTHFR activity does not affect methionine-dependency in this system.
The results of our experiment suggest that relatively methionine-dependent lymphocytes proliferate faster than methionine-independent cells in met+hcy– serum-free media. This finding is in agreement with that of Finkerstrand et al. (1997) who reported that a methionine-dependent glioma cell line (P60) proliferated at a faster rate than the methionine-independent variant (P60H) in met+hcy– media. One plausible explanation for an elevated division rate of methionine-dependent cells in met+hcy– media is that homocysteine levels are elevated due to an inefficient recycling to methionine (which we speculate to be the underlying cause of methionine-dependency). This, in turn, may cause a build up of SAM and a subsequent increase in polyamine synthesis in methionine-independent cells. Polyamines are inducers of cell proliferation (Heby, 1981). However, our data on plasma homocysteine concentrations do not appear to support the view that methionine-dependence phenotype is a cause of hyperhomocysteinemia.
Because methionine-dependency can exist in both normal and cancer cells of the same individual (Mikol and Lipkin, 1984), it seems likely that methionine-dependent cancer cells may simply develop in individuals who are originally methionine-dependent. These observations do not exclude the possibility that methionine-dependence may be a risk factor for cancer. Although our studies have shown no relationship between methionine-dependency and DNA damage, as measured by the cytokinesis-block micronucleus assay, results in serum-free medium suggested an increased rate of cell proliferation which may be a risk factor for cancer by increasing the probability of uncontrolled cell division in the early and late stages of cancer. It is possible that the consumption of methionine-rich foods (such as meat, fish and dairy) may increase the risk for cancer or increase cancer growth by a (polyamine-mediated?) stimulation of cell division. Methionine-restriction has been proposed as a method for controlling the growth of methionine-dependent tumors (or tumors in individuals with the methionine-dependent phenotype) (Breillout et al., 1990; Guo et al., 1993; Hoshiya et al., 1995).
In conclusion, we have shown that normal human lymphocytes exhibit a wide variation of methionine-dependency or abilities to proliferate in met–hcy+ media. Furthermore methionine-dependency is not related to baseline micronucleus frequency and is not determined by common mutations in MTHFR and methionine synthase. We found that methionine-dependency is related to the ability of cells to proliferate in serum-free met+hcy– media and speculate that this may be due to a higher production of growth-stimulating polyamines in methionine-dependent cells.
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Acknowledgments |
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We greatly appreciate the technical help of Carolyn Salisbury and the collection of blood by the CSIRO Clinic staff. Thank you to Ben Brinkman who performed the homocysteine analyses. Thank you to Dr Rima Rozen of Montreal Children's Hospital Research Institute for sending DNA samples for use as positive controls.
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Notes |
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3 To whom correspondence should be addressed. Email: michael.fenech{at}hsn.csiro.au
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References |
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Received on December 12, 2000; accepted on February 14, 2001.
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