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Experimental Therapeutics |
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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AGT appears to be a very widely distributed protein, and sequences are now known from at least 25 different species. Some of the microbial AGTs have been found to be very resistant to inactivation by BG (9) . Several human AGT mutants with a reduced ability to react with BG have been obtained either by site-directed mutagenesis (11, 12, 13, 14) or by screening cDNA libraries containing random insertions in the AGT sequences for the ability to protect E. coli from killing by MNNG in the presence of BG (14, 15, 16) .
Such resistant mutants are of interest for several reasons: (a) they suggest that resistance to BG therapy may arise relatively readily, and it will be necessary to obtain additional AGT inhibitors able to inactivate these resistant mutants; (b) the comparison of BG-sensitive and BG-resistant AGT mutants provides more information about the interaction of the protein and the drug that should prove valuable in designing improved inhibitors with greater potency and specificity; and (c) the dose-limiting toxicity of therapy with BG plus alkylating agents is myelosuppression, which is due to the very low level of AGT in bone marrow progenitor cells (17 , 18) . The use of a BG-resistant AGT-mutant protein for gene therapy approaches in which an AGT is expressed from a suitable vector in hematopoietic cells may overcome this toxicity and improve the chemotherapeutic index of the treatment because the tumor would remain sensitive whereas the bone marrow would be resistant (19, 20, 21, 22) .
We (12, 13, 14) and others (15 , 16) have, therefore, been attempting to produce a panel of BG-resistant mutants and to identify the major factors involved in permitting AGT to continue to work on DNA adducts but to be inactive on BG. A screening method to obtain such mutants was set up by using the ability of expressed human AGT to protect a BG-permeable E. coli strain lacking endogenous alkyltransferase (TRG8) from killing by MNNG (14) . This protection was abolished by BG. We have shown that this screen can be used to identify BG-resistant mutants in human AGT containing a random sequence inserted in place of the codons corresponding to amino acids 138–140 (14) . This screen indicated that the conversion of proline-140 to lysine (mutant P140K) produced by far the greatest resistance of any point mutation, increasing the ED50 value for inactivation by more than 12,000-fold.
Another position at which human AGT can be altered to provide resistance to BG is at glycine-160. This was discovered by the comparison of the properties of a naturally occurring variant G160R that was found in a minority of Japanese subjects (23) with wild-type AGT (13) . The G160R AGT was slightly more resistant to BG (20-fold increase in ED50), whereas the mutants G160A and G160W, which were made as controls, were found to be more sensitive than wild type (13) . The finding that G160W was sensitive to BG was later confirmed by Rafferty et al. (24) .
In the course of studies of the possible-BG resistance of an AGT-mutant population in which the codon at position 160 was replaced by NNS to produce a complete library covering replacements with all amino acids, we have now found a highly BG-resistant mutant. However, sequencing of this mutant revealed that it contained the double change Y158H/G160A. The G160A mutation was unlikely to explain the properties of this mutant because previous work had shown that G160A mutant AGT is not resistant but is slightly more sensitive than wild-type AGT (13) . Also, position 158 in AGT sequences is highly conserved with 25 of 28 known AGT sequences having tyrosine and the other three having the closely related phen-ylalanine.
We have, therefore, constructed the Y158H-mutant AGT by site-directed mutagenesis and carried out a detailed study of this and related AGT mutants. These studies show that the Y158H AGT is active in repairing DNA and is highly resistant to BG. Molecular modeling studies suggest that the charged residues on the side chains of either the P140K or the Y158H mutants may be present in the same vicinity at the AGT active site. The presence of a charged hydrophilic residue in this position may prevent the binding of the hydrophobic BG.
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MATERIALS AND METHODS |
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Plasmids for Expression of AGT.
AGT was expressed in the pUC-18 vector for selection and studies in E. coli extracts. Expression from the pQE or pIN vectors was used for purification of the recombinant protein. Plasmids pUC-AGT (14)
, pQE-AGT (13)
, pQE-P140K (14)
, pIN-Y158F, and pIN-Y158A (27)
have been described previously.
The pUC-Y158H mutant was made by PCR by using primer 1 (5'–CCGTTTTCCAGCAAGAGTCG-3') matching nt 310 to nt 432 in the pQE-AGT plasmid and primer 2 (5'–CCTTCACGGCTAGCCCTCCGGAGTGGTTGCCCACGGCTCC-3') for the mutation of the codon for tyrosine-158 to histidine (mismatch is underlined and NheI site is shown in italics). The PCR reaction was done using Pfu polymerase (Stratagene) with pQE-AGT plasmid as template under the following conditions: (a) initial denaturation for 2 min at 92°C; (b) 25 cycles of denaturation (30 s at 92°C); (c) annealing (30 s at 52°C); and (d) extension (30 s at 72°C) followed by a final extension at 72°C for 5 min. The PCR product (236 bp) was purified, digested with MluI and NheI and ligated into the pUC-inAGT2 plasmid (described below in the next section of "Materials and Methods") digested with the same enzymes to form pUC-Y158H. Plasmid pQE-Y158H was made by ligation of the 243-bp DNA containing the sequence for the Y158H mutation (obtained by digestion of pUC-Y158H with MluI and AgeI) with pQE-AGT digested with the same enzymes.
The P140K/Y158H mutant AGT was generated by using pQE-P140K and pQE-Y158H. The 1.2-kb fragment between the DraIII (at 436) and the XbaI (at 1600) restriction sites from pQE-Y158H was ligated with the 2.8-kb fragment from pQE-P140K digested with the same enzymes to form pQE-P140K/Y158H.
The P140K/Y158F mutant was generated by using the pQE-P140K and pIN-Y158F DNA constructs. The 390-bp fragment from pIN-Y158F generated by DraIII digestion (which cuts the pIN-AGT plasmid at two sites at 436 and 826 because of a site in the vector sequence) was isolated. This fragment was further digested with the AgeI restriction enzyme (at 521), dephosphorylated, and ligated with the large fragment from pQE-P140K digested with DraIII (at 436) and AgeI (at 521) to form pQE-P140K/Y158F.
The entire AGT coding region of all of the plasmids used for AGT expression and activity measurements were sequenced to ensure no secondary mutations were formed.
Construction and Screening of AGT Library with a Random Sequence Corresponding to AGT Codon 160.
A 1040-bp irrelevant inactivating sequence was inserted into the AGT cDNA in pGEMAGT2 (14)
between the DraIII (at 436) and the NheI (at 483) sites to prevent contamination of the library with wild-type AGT. The DNA insert was created by PCR using Pfu polymerase and pCM9 (28)
as template with sense primer 5'-GGAAGCTGCACAGAGTGTCGAAGGGACCGAGAAGC-3' (mismatches underlined) to create the DraIII site (shown in italics) and the antisense primer 5'-CGACTCTAGCTAGCATCCACCACC-3' (mismatches underlined) to create the NheI site (shown in italics). The PCR product was purified by the PCR Purification kit (Qiagen), digested with DraIII and NheI, and ligated into pGEM-AGT2 plasmid digested with the same enzymes to form pGEM-inAGT2. From this plasmid, the 1078-bp piece fragment between DraIII and AgeI sites was isolated and inserted into the large fragment (3 kb) from pUC-AGT plasmid digested with the same enzymes to form pUC-inAGT2. The resulting plasmid, pUC-inAGT2, was then used for insertion of the random sequences. The DNA insert containing the random sequences was created by PCR covering the AGT coding region from 420 to 540, including the DraIII site (at 436) and the AgeI site (at 521), using as the sense primer, 5'-CCGTGCCACAGAGTGGTCTGCAGCAGCGGAGCCGTGGGCAACTA-CTCCNNSGGGCTAGCCGTGAAGG-3', and as the antisense primer, 5'-GGCTTCCCCAACCGGTGGCC-3', (mismatches underlined, and restriction sites shown in italics). The PCR reaction was carried out using Pfu polymerase and pUC-AGT as the template under the same conditions as described above. The PCR product was gel-purified, digested with DraIII and AgeI enzymes, and ligated into pUC-inAGT2 digested with the same enzymes. The ligated products were used to transform XL1-Blue cells. An aliquot of the transformation mixture was plated on LB plates supplemented with 50 µg/ml ampicillin to determine the total number of plasmid-containing bacteria, which was found to be about 2.5 x 105. The remainder of the transformation mixture was amplified by growing overnight. The plasmid DNA was isolated, subjected to sequencing analysis to confirm that the randomized sequence was present, and introduced into the TRG8 strain by electroporation. An aliquot of the sample was plated to determine the efficiency of transformation (2 x 108 transformants/µg DNA), and the rest was amplified overnight in LB medium containing ampicillin and kanamycin (50 µg/ml each). Aliquots of this library were either subjected to the screening process or stored at -70°C for further use.
The screening for active and BG-resistant AGT mutants was carried out as described previously (14) using treatment with 50 µM BG for 1.5 h followed by 40 µg/ml MNNG for 0.5 h at 25°C. Crude extracts for the determination of AGT activity were prepared from the independent mutants grown overnight in 10 ml of LB medium by sonicating the bacterial pellet resuspended in 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, and 5 mM DTT for 2 min at 0°C using an ultrasonic cell disruptor model W-225-R on pulse setting 50% duty cycle. Cell debris was pelleted by centrifugation at 4°C for 15 min at 15,000 x g, and the supernatant was used to determine the AGT activity and its sensitivity to BG.
MNNG Survival Assay in the Presence of BG.
TRG8 bacteria containing plasmids expressing wild-type Y158H or Y158H/G160A AGT were grown in 5 ml of LB media containing 50 µg/ml ampicillin and 50 µg/ml kanamycin with agitation at 250 rpm in a water bath at 37°C until the A600 was 0.5. The cultures were then pelleted, washed, resuspended in 1 ml of LB media and divided into two 0.5-ml aliquots, one of which was supplemented with 250 µM BG. The tubes were agitated at 250 rpm for 1.5 h at 25°C before an additional 2 ml of media with or without BG was added to restore the original volume. After BG treatment, the cultures were exposed to MNNG (0–40 µg/ml) for 30 min. The reactions were stopped by diluting small aliquots of the bacteria cultures in M9 media on ice. The bacteria were further diluted and spread on LB plates containing 50 µg/ml ampicillin and 50 µg/ml kanamycin with or without 100 µM BG. The plates were incubated at 37°C for 16 h, and the colony numbers were determined. The percentage of survival was determined by using the colony number/ml of culture exposed to MNNG divided by the colony number/ml of culture when MNNG was absent.
Purification of AGT.
The wild type and all of the mutants except Y158F were expressed using the pQE vector, which adds a 12-amino acid sequence [MRGS (H)6GS-) to the NH2 terminus of the AGT protein, and were purified by immobilized metal affinity chromatography as described previously (13
, 14)
. The recombinant Y158F mutant protein was produced using the pIN-Y158F plasmid and was purified as described (27)
.
Determination of Effects of BG on AGT Activity.
Inactivation of the AGT activity by BG in crude bacterial extracts or purified protein preparations was measured by incubating aliquots of the protein with different concentrations of BG at 37°C for 30 min in 0.5 ml of 50 mM Tris-HCl (pH 7.6), 5 mM DTT, and 0.1 mM EDTA containing 50 µg hemocyanin and 10 µg calf thymus DNA (14)
. The residual AGT activity was determined, and the results were expressed as the percentage of the AGT activity remaining. Graphs of AGT activity remaining against inhibitor concentration were used to calculate an ED50 value representing the amount of inhibitor needed to produce a 50% loss of activity.
Measurements of the formation of [8-3H]guanine from [8-3H]BG were carried out as described previously (26) using various amounts of the purified AGT proteins in an assay buffer consisting of 0.7 µMO6-benzyl-[3H]guanine, 25 µg of calf thymus DNA, 50 mM Tris-HCl (pH 7.6), 0.1 mM EDTA, and 5 mM DTT in an assay volume of 0.25 ml.
Kinetics of Repair by AGT.
The rate constant for the AGT reaction was determined by measuring the appearance of the [3H]methylated AGT at various times using concentrations of AGT protein determined in preliminary experiments to readily measurable rates under the assay conditions (29)
. The reaction mixture (1 ml) for each time point contained AGT (4.5 x 10-10M for wild type, 2.75 x 10-9M for Y158H, 6.5 x 10-9M for P140K, 1.1 x 10-8M for P140K/Y158H or 3.4 x 10-9M for P140K/Y158F), 3.6 x 10-10MO6 -[3H]methylguanine in [3H]methylated DNA substrate and 50 µg of cold calf thymus DNA in a buffer containing 50 mM Tris-HCl (pH 7.6), 5 mM DTT, and 0.5 mM EDTA.
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RESULTS |
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and by assays using crude extracts from the TRG8 cells (Fig. 2)
.
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. Direct proof that the Y158H/G160A mutant was resistant to inactivation by BG was obtained using crude extracts from the cells and measuring AGT activity after exposure to the inhibitor (Fig. 2)
. The Y158H/G160A mutant was more than 1000 times more resistant to BG than wild type, with an ED50 value of 
180 µM as compared with 0.1 µM.
Previous studies with the purified protein have shown that the single mutation G160A does not render AGT resistant to BG and, in fact, slightly increases sensitivity to this drug (13)
. It, therefore, seemed likely that this mutation was not contributing to the resistance of the Y158H/G160A double mutant, although it might aid in maintaining the activity of the protein when the highly conserved tyrosine-158 was replaced by histidine. To test this, the single Y158H mutant AGT sequence was made and expressed in TRG8 cells. The cells expressing this protein showed similar protection from MNNG to cells expressing wild-type AGT, and this protection was not abolished by BG (Fig. 1)
. Also, the Y158H-mutant AGT in crude extracts from the TRG8 cells was actually significantly more resistant to BG inactivation than the double mutant (Fig. 2)
.
This resistance was confirmed using AGT purified to homogeneity (Fig. 3
and Table 1
). The Y158H mutant AGT was expressed using the pQE vector, which places a (His)6-tagged sequence at the NH2 terminus. The protein was then purified using immobilized metal affinity chromatography. As shown in Fig. 3
, the ED50 for inactivation by BG of the Y158H AGT was greatly increased, whereas the Y158F mutation had little significant effect on the reaction with BG. The ED50 value for Y158H was increased by more than 6000-fold over wild-type AGT from 0.1 µM to 620 µM(Table 1)
.
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. The presence of a positive charge in this location may, therefore, strongly discourage the binding of the hydrophobic BG.
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. Measurement of the rate of guanine production when AGT acts on BG provides a more sensitive method for determining the interaction of AGT with BG (26)
. Such studies were, therefore, carried out using [8-3H]BG as a substrate in a 20 min reaction. The production of [8-3H]guanine by the mutants P140K, Y158H and Y158F was consistent with the ED50 values (Table 1)
. As reported previously (14)
, the formation of [8-3H]guanine was greatly reduced by the P140K mutation. The Y158H mutation was slightly less effective but it also greatly reduced [8-3H]guanine formation, whereas the Y158F mutation had only a small effect (Table 1)
. The P140K/Y158F and P140K/Y158H double mutants had a highly impaired ability to convert [8-3H]BG to [8-3H]guanine but P140K/Y158F was actually more reactive than P140K, whereas P140K/Y158H gave no detect-able guanine formation even when 0.6 mg of AGT protein was used (Table 1)
.
Although the Y158H and P140K mutations clearly did not prevent the ability of AGT to repair methylated DNA, they did produce a significant effect on the rate of the reaction. As shown in Table 1
, there was an approximately 10-fold reduction in the rate constant for DNA repair. These reductions may reflect the distortion at the active site. The combination of the P140K mutation with the Y158H mutation that may produce an even greater distortion reduced the rate constant by a greater amount (40-fold) but the combination P140K/Y158F gave a smaller reduction of only 4-fold. This may indicate that the loss of the hydroxyl group from the side chain of residue 158 may allow the lysine inserted at position 140 to be accommodated with less of an alteration in the structure of the active site.
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DISCUSSION |
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, these results should not be taken to indicate that the double mutant is more active. The observed difference may be an experimental artifact because the cells expressing the double mutant were those surviving the treatment with BG plus MNNG. These cells may have acquired additional mutations in the bacterial genome during the selection procedure, and they grew more slowly than the original untreated TRG8 cells in which the wild type and the Y158H mutants were expressed. This difference in growth rate or other effects of unknown mutations could contribute to the difference observed. Our results show that, although the position in all of the alkyltransferases equivalent to 158 in the human AGT is highly conserved in all of the known AGTs and is restricted to tyrosine or occasionally phenylalanine, the substitution with histidine also gives rise to an active protein. It is possible that these amino acids are the only ones tolerated at this position because mutant Y158A was inactive (27) , and no AGTs with other alterations at position 158 have been found in screens of mutated AGT libraries. The ability of histidine to replace tyrosine-158 and to provide an active AGT is also suggested by recent results of the screening using BG and MNNG for the selection of plasmid libraries in which random mutagenesis was used for substitutions of positions 150–172 of AGT (16) . All of the BG-resistant mutants found had multiple substitutions with 3–5 amino acids differing from wild type, but 5 of 8 of these mutants had the Y158H change. Our results suggest that this alteration was the major reason for the resistance of these mutants to BG.
Several microbial AGTs including the Ada-C protein from E. coli are virtually unaffected by BG (9) . Studies by site-directed mutagenesis and comparison with the known crystal structure for this protein (30 , 31) have indicated that the resistance of the E. coli Ada-C AGT is due to a steric restriction of the size at the active site that prevents the binding of the bulky BG (32 , 33) . This restriction is caused by the combination of the absence of a proline and the presence of a tryptophan residue in the active site pocket. The mutation of both of these residues to form proline and alanine, respectively, renders Ada-C able to react with BG (32 , 33) .
The inability of the first reported BG-resistant mutants of the human AGT (P140A and G156A) to react well with this drug also seems to be due to steric factors inasmuch as these alterations would be expected to reduce the size of the active site pocket in the human AGT (11 , 12) . However, the discovery that the G160R mutant was also resistant, whereas G160A and G160W were not (and were actually more sensitive), suggested that another class of resistant mutants might be formed by the presence of a hydrophilic group in the active site pocket (13) .
The finding that the Y158H-mutant AGT is highly resistant to inactivation by BG (ED50 of 620 µM) provides strong support for this hypothesis. Its resistance can be explained by the presence of a charged residue in the active-site pocket that discourages the binding of the hydrophobic BG. The other two single amino acid changes that have the largest effect in rendering AGT resistant to BG are P140K (ED50 of > 1200 µM) and P140R (ED50 of 190 µM; Ref. 14 ). These changes are also likely to lead to placing a charged residue in the active site and may also influence the reaction by the steric effect caused by replacing the proline described above.
The combination of the P140K and Y158H mutations forming mutant P140K/Y158H rendered human AGT totally unable to react with BG. The limit of detection in the assays shown in Table 1
is such that the reduction in rate of reaction of this double mutant when compared with wild type is by a factor of more than 105. This P140K/Y158H mutant was considerably less active with BG than the single P140K mutant. Although a small part of this reduction may be due to the difference in rate constant between these two mutants, this was altered by only a factor of 3.6, whereas the reduction in the rate of guanine formation from BG was more than 20-fold (Table 1)
. In contrast, the increased ability of the P140K/Y158F to react with BG when compared with P140K alone may be due solely to the alteration in the rate constant.
Although both the P140K- and the Y158H-mutated AGT proteins have a significant reduction in the rate constant for the repair of methylated DNA, this does not prevent them from being highly effective in protecting E. coli from killing by MNNG (Fig. 1
and reference (14)
) or, in the case of P140K, from protecting mammalian cells from the therapeutic agent BCNU (34)
. This is probably due to the very rapid rate of repair by the wild-type AGT. A moderate reduction in this rate, therefore, has little physiological effect. In studies with other mutants of AGT in the DNA binding domain that are more severely compromised in their activity, a greater than 35-fold reduction was required before any effect was observed. Even mutant Y114A, which had almost a 1000-fold reduction in rate constant, provided some protection (29)
.
The protection from killing by alkylating agents afforded by mutants Y158H and P140K was not reduced by BG even at concentrations far in excess of those likely to be achievable in patients treated with this drug. Therefore, these mutants are excellent choices for gene therapy approaches in which the expression of a BG-resistant mutant AGT in hematopoietic progenitor cells is used to protect the bone marrow and improve the therapeutic index of such therapy.
Conversely, the relatively facile production of BG-resistant mutants that is revealed in these and other experiments suggests that the formation or selection for such forms of AGT in tumor cells during therapy may be a significant problem although these forms have lower-than-wild-type-AGT activity. This focuses attention on the development of novel inhibitors that could be used to inactivate such forms. The use of short single-stranded oligodeoxynucleotides containing an O6-benzyldeoxyguanosine may be useful in this regard because the binding of these to the AGT-active site involves the DNA binding domain and is much stronger than the binding of the free base BG (35) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by National Cancer Institute Grants CA-71976, CA-57725, and CA-18137. J. Leitao was the recipient of fellowships for mobility granted by Foundation Calouste Gulbenkian and Luso-American Development Foundation. 
2 Present address: Unidade de Ciencias e Tecnologias Agrarias, Universidade do Algarve, Campus de Gambias, 8000 Faro, Portugal.
3 To whom requests for reprint should be addressed at Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033. Phone: (717) 531-8152; Fax: (717) 531-5157; E-mail: aep1{at}psu.edu
4 The abbreviations used are: AGT, O6-alkylguanine-DNA alkyltransferase; BG, O6-benzylguanine; ED50, concentration of BG needed to reduce the alkyltransferase activity by 50% in a 30 min incubation at 37°C; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; LB, Luria-Bertani medium.
Received 9/24/98. Accepted 2/ 2/99.
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, •), mutant Y158H (
, 
), and Y158H/G160A (
, 
).
