Laboratory of Pharmacology, Istituto Dermopatico Dell'Immacolata (IDI-IRCCS), Via dei Monti di Creta 104, 00167 Rome,
1 Department of Neuroscience, Pharmacology and Medical Oncology Section and
2 Department of Public Health and Cell Biology, University of Rome `Tor Vergata', Via di Tor Vergata 135, 00133 Rome, Italy and
3 Institute for Medical Radiobiology, University of Zurich, August Forel-Strasse 7, CH-8029 Zurich, Switzerland
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Abstract |
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Abbreviations: BG, O6-benzylguanine; BSA, bovine serum albumin; CM, complete medium; DHFR, dihydrofolate reductase; IDLs, insertion/ deletion loops; MMR, mismatch repair; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTX, methotrexate; O6-G, O6-guanine; OGAT, O6-alkylguanine-DNA alkyltransferase; O6-MeG, O6-methylguanine; PBS, phosphate buffered saline; PI, propidium iodide; 6-TG, 6-thioguanine; TMZ, temozolomide, 8-carbamoyl-3-methyl-imidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one.
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Introduction |
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Biochemical and genetic studies in human cells have defined at least five genes whose products play key roles in mismatch repair: hMSH2, hMSH3, hMSH6, hMLH1 and hPMS2 (1,2). The protein complex hMutS, a heterodimer of hMSH2 and hMSH6, or hMutSß, consisting of hMSH2 and hMSH3, initially recognize and bind mismatched DNA. After this step, a heterodimer of hMLH1 and PMS2, termed hMutL
, interacts with the DNA-bound hMutS
or hMutSß to initiate the repair process (1,2).
hMutS and hMutSß possess distinct substrate specificities. The hMutS
complex binds to base/base mismatches and one, two and three nucleotide insertion/deletion loops (IDLs), while the hMutSß heterodimer displays little or no affinity for base/base mismatches, but binds to multiple base IDL-type misalignments with high efficiency (13).
Although the MMR system has evolved for the correction of replication errors, it is also implicated in the recognition of other types of DNA damage and in the triggering of events leading to cell death. Indeed, in the past few years, deficiency in MMR was shown to be linked with tumour cell resistance to a number of chemotherapeutic agents (for review, see ref. 4). Inactivation of either hMSH2 or hMLH1 has been associated with high levels of resistance to 6-thioguanine (6-TG) (4,5) and O6-guanine (O6-G)-methylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (4) or 8-carbamoyl-3-methyl-imidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one (temozolomide, TMZ) (6,7), as well as with a moderate degree of resistance to cisplatin (4,8), carboplatin (4,9) and etoposide (4,9,10). Loss of hMLH1 has been shown to confer resistance to doxorubicin (4,9). Mutations in hMSH6 have been reported to decrease cell sensitivity to O6-G-methylating agents (4,11,12), 6-TG (1), cisplatin (8), but not to etoposide (11,13). Little is known about the effect of mutations in hMSH3 and hPMS2 on drug sensitivity. So far, absence of hPMS2 expression has been associated with resistance to MNNG (4), cisplatin and carboplatin (14), whereas no changes in sensitivity to cisplatin (8) or MNNG (15) have been found in cells harbouring mutations in hMSH3.
Recently it has been demonstrated that mismatch repair deficiency can arise as a result of imbalance in the relative amounts of hMSH3 and hMSH6 proteins (16,17). The hMSH3 gene is divergently transcribed from the dihydrofolate reductase (DHFR) promoter (18). Cells resistant to methotrexate (MTX) as a result of amplification of the DHFR locus, also overexpress the hMSH3 protein, which then displaces hMSH6 from its complex with hMSH2. Cells thus became defective in the repair of base/base mismatches and display a mutator phenotype (16,17). However, they retain the ability to correct IDLs.
Methotrexate is widely used for the treatment of human malignancies (19). It is also used in the treatment of some autoimmune diseases and for the prevention of graft-versus-host diseases in transplant patients (19). Resistance to MTX caused by amplification of the DHFR gene is frequently observed in tumour cells (20). It appears, therefore, of clinical interest to establish whether the associated co-amplification of the hMSH3 locus confers cross-resistance to drugs unrelated to MTX, but whose cytotoxic activity could be modulated by the MMR status of the cell.
The chemoresistance pattern of human tumour cells overexpressing the hMSH3 gene has not been investigated so far. In this study we evaluated the ability of several chemotherapeutic agents to bring about growth inhibition, apoptosis and chromosome aberrations in the MMR-proficient cell line HL-60 and in its MTX-resistant subline HL-60R, which overexpresses the hMSH3 gene and is deficient in the repair of base/base mismatches (16,17).
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Materials and methods |
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The cell lines were cultured at 37°C in 5% CO2 humidified atmosphere and maintained in RPMI-1640 (Hyclone Europe, Cramlington, UK) supplemented with 20% heat-inactivated (56°C, 30 min) fetal calf serum (Hyclone, Logan, UT), 2 mM L-glutamine and antibiotics (Gibco BRL, Life Technologies, Paisley, Scotland) (referred to as complete medium, CM). Once a month, the MTX-resistant line was cultured in the presence of 1 µM MTX for 1 week. Overexpression of the hMSH3 protein in HL-60R cells was periodically verified by Western blot analysis (data not shown).
Drugs and reagents
Temozolomide was kindly provided by Schering-Plough Research Institute (Kenilworth, NJ). Etoposide, 6-TG, O6-benzylguanine (BG), colchicine and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) were purchased from Sigma (St Louis, MO). MNNG was obtained from Aldrich (Milan, Italy). Cisplatin (prontoplatamine, 500 µg/ml in saline, pH 35) was purchased from Pharmacia & Upjohn (Milan, Italy). Doxorubicin (adriblastina) was obtained from Pharmacia (Milan, Italy).
Etoposide and MNNG were dissolved in dimethyl sulfoxide. BG and 6-TG were dissolved in ethanol and 0.1 N NaOH, respectively. Colchicine and doxorubicin were dissolved in saline. All these reagents were stored as stock solutions at 80°C, and diluted in CM just prior to use. The final concentrations of dimethyl sulfoxide, ethanol or NaOH did not affect cell growth (data not shown). Temozolomide was always prepared fresh by dissolving the drug in CM. MTT was dissolved at a concentration of 5 mg/ml in phosphate buffered saline (PBS) and stored at 4°C.
Reagents for agarose gel electrophoresis were all purchased from Bio-Rad (Hercules, CA).
Evaluation of cell chemosensitivity by MTT assay
HL-60 and HL-60R cells were suspended in CM at a concentration of 6x104 cells/ml and dispensed in 50 µl aliquots into flat-bottom 96-well plates (Falcon; Becton & Dickinson, Frankein Lakes, NJ). Graded amounts of each drug were then added to the wells in 50 µl CM and the plates were incubated at 37°C in a 5% CO2 humidified atmosphere for 72 h. Four replica wells were used for controls and each drug concentration.
The effects of MNNG and TMZ were also evaluated in the presence of the OGAT inhibitor BG (22) to prevent repair of the methyl adducts at the O6-G. To this end, cells were suspended in CM containing 10 µM BG, plated as described above and incubated at 37°C for 2 h. Temozolomide or MNNG were then added in 50 µl CM and the plates were maintained at 37°C for 72 h. Cells were therefore also exposed to 5 µM BG during the entire period of drug treatment. Under these conditions a complete abrogation of OGAT activity was obtained before drug treatment and up to the end of the assay (data not shown). Control groups were either untreated or treated with BG alone.
The MTT assay was performed as previously described (23). Briefly, after 72 h of culture, 0.1 mg MTT (in 20 µl PBS) was added to each well and cells were incubated at 37°C for 4 h. Cells were then lysed with buffer (0.1 ml/well) containing 20% SDS and 50% N,N-dimethyl formamide, pH 4.7. After an overnight incubation, the absorbance was read at 595 nm using a 3550-UV microplate reader (Bio-Rad).
Cell sensitivity to drug treatment was expressed in terms of IC50 (drug concentration producing 50% inhibition of cell growth, calculated on the regression line in which absorbance values at 595 nm were plotted against the logarithm of drug concentration).
Evaluation of cell chemosensitivity by cell count
HL-60 and HL-60R cells were suspended in CM at the final concentration of 2x105 cells/ml and dispensed in 1 ml aliquots in 24-well plates (Falcon). Graded concentrations of the drugs under investigation were then added to the wells in 1 ml CM and the cells were cultured at 37°C in a 5% CO2 humidified atmosphere for 72 h. Cell growth was evaluated in terms of viable cell count at the end of the incubation period. Cells were manually counted using a haemocytometer and cell viability was determined by trypan blue exclusion test. All determinations were made in duplicate. The effects of TMZ were evaluated in the presence of BG, which was added at the concentration of 10 µM 2 h before addition of the drug, and maintained in culture at a concentration of 5 µM. Control groups were either untreated or treated with BG alone.
Data were expressed in terms of percentage of cell growth of drug-treated groups with respect to controls. IC50 values were calculated on the regression lines in which the number of cells was plotted against the logarithm of drug concentration.
Evaluation of drug-induced chromosome aberrations
HL-60 and HL-60R cells were suspended in CM at the final concentration of 2x105 cells/ml and dispensed in 2.5 ml aliquots in 6-well plates (Falcon). TMZ, MNNG, cisplatin or etoposide were then added to the wells in 2.5 ml CM and the cells were cultured at 37°C in a 5% CO2 humidified atmosphere for 48 h. The cultures were exposed for the last 2 h of incubation to 0.8 µg/ml colchicine. In the case of TMZ and MNNG treatments, BG was added to the cell suspensions at a concentration of 10 µM 2 h before the drug and maintained in culture at a concentration of 5 µM. Control groups were either untreated or treated with BG alone.
At the end of the incubation period, cells were harvested, treated for 15 min with a hypotonic solution (0.075 µM KCl) and fixed by 3:1 (v/v) methanol/acetic acid, before dropping them onto clean wet slides. Slides were then stained with a 5% Giemsa solution in Sorensen buffer (pH 6.8). 100 metaphases, from at least two independent cultures for controls and each drug-treated group, were scored to evaluate the number of chromosome aberrations (chromatid and chromosome breaks) per cell, and the number of cells with chromosome aberrations (aberrant cells). Chromatid (i.e. triradials) and chromosome (i.e. dicentrics) rearrangements were counted as two chromatid and two chromosome breaks, respectively. No evaluation of sister chromatid exchanges was performed since neither cell lines incorporated 5-bromo-2'-deoxyuridine into their DNA (data not shown).
Assessment of apoptosis by flow cytometric analysis
Cells from control or drug-treated cultures were harvested by centrifugation, washed with PBS and fixed with 70% ethanol at 20°C for 18 h. They were then centrifuged, resuspended in 1 ml hypotonic solution containing 50 µg/ml propidium iodide (PI), 0.1% sodium citrate, 0.1% Triton-X and 10 µg/ml RNAse and incubated in the dark at room temperature for 1 h. Propidium iodide fluorescence was measured on a linear scale using a FACScan flow cytometer (Becton & Dickinson, San Jose, CA). Data from 2x104 cells were recorded and analysed using CellQuest software (Becton & Dickinson). Data collection was gated utilizing forward and side light scatter to exclude cell debris and cell aggregates. Apoptotic cells were determined by their hypochromic, sub-G1 staining profiles (24).
Assessment of apoptosis by DNA fragmentation assay
Cells were collected by centrifugation, washed twice in PBS, resuspended (5x106 cells) in 0.2 ml 10 mM TrisHCl, pH 8.6, 1.5 mM MgCl2, 140 mM NaCl and 0.5% NP-40. Samples were incubated for 5 min on ice and subsequently microfuged for 5 min at 14 000 r.p.m. Supernatants were removed and incubated in the presence of 0.25 mg/ml DNAse-free, RNAse A at 37°C for 1 h. After addition of 0.2 ml 2x proteinase K buffer (0.2 M TrisHCl, pH 8.0, 25 mM EDTA, 0.3 M NaCl and 2% SDS) samples were incubated for 30 min at 37°C in the presence of 50 µg/ml proteinase K. Low molecular weight DNA was then extracted once with phenol [buffered with 24:24:1 (v/v/v) 0.1 M TrisHCl pH 7.4/chloroform/isoamyl alcohol] and precipitated for 24 h in the presence of 0.1 M sodium acetate, pH 5.2, with 1 vol isopropanol. DNA precipitates were recovered by centrifugation at 14 000 r.p.m. using a refrigerated microfuge, and analysed by electrophoresis in 2% agarose gel containing 0.5 µg/ml ethidium bromide. DNA was visualized and photographed using a UV transilluminator and a Polaroid camera set-up.
Statistical analysis
Statistical analysis was performed, taking into account the results of all independent experiments available. Mean and standard error (SE) of the percentages relative to cell growth and apoptosis were obtained after `angular transformation' of the calculated percentages, in order to process normally-distributed data. Therefore, statistical analysis, performed according to Student's t-test, was carried out by using transformed data.
When the concentrationeffect relationship was considered, regression line analysis was carried out on the absorbance values at 595 nm or the number of cells versus the logarithm of drug concentration. Thereafter, the IC50 values relative to the drugs under investigation were determined for HL-60 and HL-60R cells in each separate experiment. Differences between IC50 relative to parental HL-60 line and those relative to HL-60R subline were then subjected to statistical analysis according to Student's `pair t-test'.
The statistical analysis of HL-60 and HL-60R cell sensitivity to drug-induced chromosome aberrations was performed on the number of aberrant cells, and not on the number of aberrations per cell, because of the variability in chromosome numbers in both lines and the presence of the large amplified region in the HL-60R cell line. The normal standardized deviate was calculated according to the formula
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where n1 and n2 are the mean values of cells containing aberrations, considered as absolute numbers on 100 cells. Probability values (P) were then calculated according to `U' distribution tables.
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Results |
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The results illustrated in Table I refer to drug-induced growth inhibition evaluated by the MTT assay. Cell sensitivity to each drug is expressed in terms of IC50. The data show that, in comparison to the MMR-deficient HL-60R cell line, the MMR-proficient HL-60 cells were more susceptible to the inhibitory effects of MNNG (2- and 4-fold in the absence and presence of BG, respectively), TMZ (1.3- and 5-fold in the absence and presence of BG, respectively) and 6-TG (about 2-fold). In contrast, there was no evidence of differential cytotoxicity for doxorubicin, cisplatin and etoposide.
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To evaluate sensitivity of HL-60 and HL-60R cell lines to drug-induced chromosome aberrations, the cells were treated with MNNG (2 and 8 µM), TMZ (100 and 400 µM), cisplatin (0.5 µM) or etoposide (0.05 µM) and then the number of chromosome aberrations per cell, as well as the number of aberrant cells were assessed. These drug concentrations were chosen in the range of the drug IC50 values observed for the two lines in cell growth inhibition assays.
The results illustrated in Table II show that the numbers of aberrant cells and chromosome aberrations per cell in the untreated cultures of the two lines were similar. Moreover, there was no increased chromosomal fragility in the HL-60R strain in the chromosomes containing the amplified region (data not shown). When the cells were exposed to MNNG or TMZ (in the presence of BG) a higher number of chromosome aberrations per cell and of aberrant cells was observed in the hMutS
-proficient line with respect to its hMutS
-deficient counterpart (Table II
). Statistical analysis performed on the number of aberrant cells showed that the observed differences were statistically significant at all drug concentrations tested. On the other hand, the two lines were not differentially susceptible to the clastogenic effects of cisplatin or etoposide (Table II
).
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Discussion |
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The clastogenic effects of O6-G-methylating agents also appear to be linked with the attempts of the MMR system to repair base pairs containing O6-MeG. Defects in hMutS have been associated with reduced cell susceptibility to chromosome aberrations induced by MNNG and MNU (25). In agreement with these findings, our present data show that upon treatment with MNNG or TMZ (both tested in the presence of BG), a higher number of chromosome aberrations occurred in HL-60 cells with respect to the HL-60R cell line (Table II
), indicating that MMR is involved in the generation of this kind of lesion.
That a functional hMutS complex is absolutely required for the processing of base pairs containing methylated nucleotides was further supported by our finding that the HL-60R cells were also more resistant than parental cells to 6-TG (Table I
). This base analogue is incorporated into DNA and successively methylated at the S6 position by endogenous S-adenosylmethionine. DNA replication gives rise to S6-methyl-TG/T and S6-methyl-TG/C mispairs, which are assumed, like O6-MeG/T and O6-MeG/C mispairs, to be recognized by hMutS
and thus subjected to `futile' processing by MMR, which eventually results in cell death (26).
Mismatch repair defects have been linked also with resistance to cisplatin. The hMutS complex and hMSH2 alone were reported to bind cisplatin adducts in DNA (2730) and increased resistance to this drug has been documented in several cell lines harbouring mutations in hMSH2, hMSH6, hMLH1 or PSM2 (4,8,14), but not hMSH3 (8). On this basis it has been suggested that both hMutS
and hMutL
, but not hMutSß, are required for the lethal processing of cisplatin adducts by the MMR. We were thus somewhat surprised to find that this drug induced similar levels of growth inhibition (Table I
and Figure 1
), chromosome aberrations (Table II
) and apoptosis (Table III
, Figures 2 and 3
) in both HL-60 and HL-60R cells. One hypothesis that might explain our results is that hMutSß recognizes cisplatin adducts as efficiently as hMutS
. Although hMutSß is usually much less abundant than hMutS
(16,17,31), in cells overexpressing hMSH3 the concentration of the former heterodimer is substantially increased (16, 17). Thus, if the hMutSß heterodimer were able to recognize cisplatin adducts, it could compensate for the loss of hMutS
. However, even though hMutSß appears to be capable of recognizing DNA damage induced by bulky chemical carcinogens as efficiently as hMutS
(32), the ability of hMutSß to bind cisplatin intrastrand cross-links has not been tested to date.
Resistance to cisplatin is multifactorial and can be due to decreased drug accumulation, inactivation of the drug by thiol compounds, an increase in metallothionein levels and accelerated DNA repair (33,34). Moreover, HL-60 and HL-60R cell lines do not express p53 (35), and the latter cell line has a mutator phenotype. Thus, although unlikely, we cannot rule out the possibility that HL-60R cells might have acquired changes in cellular factors that regulate sensitivity to cisplatin and are thus able to counteract the effects of hMutS deficiency, or that the absence of p53 in both cell lines outweighs the influence of the different MMR status on drug sensitivity.
In the present study, we also failed to detect differential susceptibility of HL-60 and HL-60R cells to growth inhibition induced by etoposide and doxorubicin (Table I and Figure 1
). Moreover, both lines were also equally susceptible to the clastogenic and apoptotic effects of etoposide (Tables II and III
; Figure 2
). Although it is reasonable to hypothesize that inactivation of hMSH6 itself may not be sufficient to confer resistance to etoposide and doxorubicin, as discussed for cisplatin, we cannot ignore the fact that the absence of p53, or the presence of additional molecular mechanisms of resistance (for review, see refs 36 and 37), might override the effects of MMR status on HL-60 and HL-60R cell sensitivity to these agents.
In conclusion, our data demonstrate that hMSH3 overexpression induces a significant increase in cell resistance to the cytotoxic effects of O6-G methylating agents and 6-TG, as previously described for mutations in the genes hMSH2, hMSH6, hMLH1 and hPMS2. This finding has clinical relevance because MTX is routinely employed in the treatment of childhood acute lymphocytic leukemia, as well as other tumours, such as breast cancer. Amplification of the DHFR locus under MTX-selective pressure may indeed lead to a rapid appearance of a multi-drug-resistant phenotype in the tumour. It must, however, be taken into consideration that the degree of DHFR locus amplification in tumours may be limited to two to four extra copies. Although it is possible that even a small increase in hMSH3 levels could lower mismatch repair efficiency, further investigations are required to establish the pattern of drug resistance in tumours with such a moderate degree of hMSH3 amplification.
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Notes |
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Acknowledgments |
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References |
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