Kinetic Studies of DNA Interstrand Crosslink by Nitrogen Mustard and Phenylalanine Mustard Margaret I
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Rochester Institute of Technology RIT Scholar Works Theses Thesis/Dissertation Collections 10-1-1990 Kinetic studies of DNA interstrand crosslink by nitrogen mustard and phenylalanine mustard Margaret I. Kaminsky Follow this and additional works at: http://scholarworks.rit.edu/theses Recommended Citation Kaminsky, Margaret I., "Kinetic studies of DNA interstrand crosslink by nitrogen mustard and phenylalanine mustard" (1990). Thesis. Rochester Institute of Technology. Accessed from This Thesis is brought to you for free and open access by the Thesis/Dissertation Collections at RIT Scholar Works. It has been accepted for inclusion in Theses by an authorized administrator of RIT Scholar Works. For more information, please contact [email protected]. Kinetic Studies of DNA Interstrand Crosslinking by Nitrogen Mustard and Phenylalanine Mustard Margaret I. Kaminsky Thesis submitted in partial fulfLllment ofthe requirements for the degree ofMasters in Science. Approved by: Name Illegible Project Advisor Gerald A. Takacs Department Head Name Illegible Library October, 1990 Rochester Institute ofTechnology Rochester, New York 14623 Department ofChemistry Title ofThesis: Kinetic Studies ofDNA Interstrand Crosslinking by Nitrogen Mustard and Phenylalanine Mustard I, Margaret I. Kaminsky, hereby grant pennission to the Wallace Memorial Library of RIT to reproduce my thesis in whole or in part. Any reproduction will not be for commercial use or profit. Date: e--' IJ ' ~ Signature: I { I"..- Abstract Phenylalanine mustard (PAM) and nitrogen mustard (HN2) are bifunctional alkylating agants which covalently crosslink DNA. Their crosslinking ability forms the basis of their usefulness as anti-cancer drugs since crosslinked DNA cannot replicate and thus the cancers cells cannot reproduce. Despite apparent similarities, the two drugs are known to have important differences. PAM is more effective against cancer and has fewer side effects. In reactions with cell cultures, PAM produces crosslinks more slowly, but the crosslinks are more persistant The experiments reported here offer kinetic explanations for the differences. In vitro time-dependent crosslinking reaction profiles for HN2 and PAM produced using an alkaline ethidium bromide assay for crosslinked DNA are virtually identical to what has been reported in vivo using an alkaline elution method. This suggests that the differences between the two drugs can be explained by purely chemical processes. In particular, the difference in the persistence of PAM and HN2 crosslinks in vivo does not require differences in the rate of enzymatic repair of the two types of lesions since loss of crosslinks in vitro follows the same schedule. A novel modification of the ethidium bromide assay reveals that PAM and HN2 have similar solution stabilities in neutral sodium phosphate buffer. This rules out the possibility that the pharmocological differences between PAM and HN2 are attributable to differential solution stabilities. The dependence of the rate of the crosslinking reaction on drug concentration is linear for both drugs. This confirms previous in vivo studies which suggested that the crosslinking reaction is pseudo first order for drug concentration. The dependence of the reaction rate on the temperature shows that PAM and HN2 have similar activation energies for the crosslinking reaction but PAM has a less favorable steric factor as predicted based on PAM's bulky phenylalanine portion. The crosslinked products for PAM and HN2 were isolated and their rates of decomposition were shown to be first order. PAM crosslinks have a longer half-life and a larger portion of PAM's crosslinks resist decay. These observations may be sufficient to explain the differences between PAM's and HN2's reaction profiles in vivo and in vitro and may be responsible for PAM's greater therapeutic value. A model is proposed to account for PAM's longer half-life and greater fraction of decay-resistant crosslinks. Table of Contents Introduction 1 General Experimental Methods 3 Theory 3 Solutions Preparations 6 Phosphate Buffers 6 Ethidium Bromide Assay Solution 6 Ethidium Bromide Fluorescence Standard 7 DNA Solutions 7 PAM and HN2 Solutions 7 Fluorimeter Parameters 8 Denaturation Parameters 9 Calculation of Percent Crosslinking 9 Chapter 1: Reaction Profiles 11 Introduction 11 Procedures 15 DNA-Drug Reactions 15 Results 16 PAM 16 HN2 18 Discussion 21 Comparison of PAM and HN-2 Profiles 21 Comparison of Experimental Data to the Literature 21 Cellular Processes Which Affect PAM's Rate and Extent of Crosslinking 25 Transport 25 Influence of Endogenous Thiols 25 11 Importance of Loss of Crosslinks 27 Enzymatic Repair and Cell Susceptibility to PAM and HN2 27 Summary of Profiles of PAM and HN2 with Calf Thymus DNA 30 Chapter 2: Drug Solution Stabilities 32 Introduction 32 Procedures for Determining Drug Solution Stabilities 33 DNA-Drug Reactions 33 Calculation of Drug Solution Half Lives 33 Results 35 PAM 35 HN2 39 Discussion 43 Comparison of PAM and HN2 Solution Stability 43 PAM and HN-2 Decomposition in the Literature 43 Significance of New Technique to Measure Drug Activity 47 Summary of PAM and HN2 Solution Stability 49 Chapter 3: Influence of Drug Concentration on Reaction Rate 50 Introduction 50 Procedures 51 Reaction Procedures 51 Analysis of Data 51 Results 52 PAM 52 HN2 59 Discussion 66 Comparison of PAM and HN2 66 Linearity of Initial Rate Versus Drug Concentration Plot 69 Ill Proposed Reaction Mechanisms in the Literature 69 Summary of Concentration Dependence 73 Chapter 4: Influence of Temperature on Reaction Rate 74 Introduction 74 Procedures 76 Results 77 PAM 77 HN2 80 Discussion 84 Comparison of PAM and HN-2 Temperature Dependence 84 Mechanisms in the Literature Revisited 85 Summary 86 Chapter 5: Loss of Crosslinking 87 Introduction 87 Procedures 88 Exclusion Chromatography Separation of Unreacted Drugs 88 Closed Circular DNA Ethidium Bromide Assay for Strand Breaks 89 Results 91 PAM 91 HN2 93 Discussion 96 No Lag Time Seen for Crosslinking Step 96 Unimolecular Decay 97 Decay-Resistant Crosslinks 97 Is the Apparent Loss of Crosslinking an Artifact of Nicking? 99 Depurination versus Opening and the Fate of DNA Interstrand Crosslinks 101 IV Summary 107 References 1 08 iv V List of Figures and Tables Figure I-lStructural Formulas for Nitrogen Mustards 2 Figure M-l Structure of Ethidium Bromide 4 Figure M-2 Schematic for Ethidium Bromide Assay for Crosslinked DNA 5 Figure 1-1 Chart 5 from Ross etal (1978) 12 Figure 1-2 Figure 3b from Hansson et al (1987) 13 Figure 1-3 PAM Time-Dependent DNA Crosslinking Reaction Profile [Drug]/[P]=0.35, 37 oC, MK1-54 17 Figure 1-4 HN-2 Time-Dependent DNA Crosslinking Reaction Profile, Trial 1. [Drug]/[P]=0.37 , 37 oC, MK1-34 19 Figure 1-5 HN-2 Time-Dependent DNA Crosslinking Reaction Profile, Trial 2. [Drug]/[P]=0.37, 37 oC, MK1-128 20 Table 1-1 Comparison of Profile Values for HN2 23 Table 1-2 Comparison of Profile Values for PAM 24 Figure 2-1 PAM hydrolysis at 37oC, Trial 1, [Drug]/[P]=.5, RG1-133 36 Figure 2-2 PAM hydrolysis, 37oC , Trial 2, [Drug]/[P]=0.1, RG2-145 37 Figure 2-3 PAM hydrolysis at 47oC, [Drug]/[P]=1, t-half=70 min. (6 points), MK1-40 38 Figure 2-4 HN2 hydrolysis at 37oC, Trial 1, [Drug]/[P]=1 with [P]=0.16 mM, RG1-57 40 Figure 2-5 HN2 hydrolysis at 37oC, Trial 2, [Drug]/[P]=1 with [P]=.16 mM, RG1-67 41 Figure 2-6 HN2 hydrolysis at 47 oC, [Drug]/[P]=1 with [P]=.15mM 42 Figure 2-7 Schematic of PAM Decomposition in the Presence of Chloride Ion et al 45 (after Chang ,1979) Figure 2-8 Schematic of Disappearance of Active Drug Intermediate 48 VI Figure 3-1 Initial Rate of DNA Crosslinking by PAM at 37oC. [Drug]/[P]=0. 1 with [P]=0. 16 millimolar, Trial 1 53 Figure 3-2 Initial Rate of DNA Crosslinking by PAM at 37 Degrees Celsius. [Drug]/[P]=0.1 with [P]=0.16 millimolar, Trial 2. Initial Slope (20 minutes) is 0.19% per minute plus or minus 0.06 % per minute. MK1-62 54 Figure 3-3 Initial Rate of DNA Crosslinking by PAM at 37oC. [Drug]/[P]=0.19 with [P]=0.16 millimolar 55 Figure 3-4 Initial Rate of DNA Crosslinking by PAM at 37oC. [Drug]/[P]=0.35 with [P]=0.16 millimolar 56 Figure 3-5 Initial Rate of DNA Crosslinking by PAM at 37oC. [Drug]/[P]=0.5 with [P]=0.16 millimolar 57 Figure 3-6 Summary of Dependence of Initial Cross-Link Rate on PAM Concentration at 37oC 58 Figure 3-7 Initial Rate of DNA Crosslinking by HN-2 at 37oC. [Drug]/[P]=0.1 with [P]=0.16 millimolar 60 Figure 3-8 Initial Rate of DNA Crosslinking by HN-2 at 37oC. [Drug]/[P]=0.2 with [P]=0.16 millimolar 61 Figure 3-9 Initial Rate of DNA Crosslinking by HN-2 at 37oC. [Drug]/[P]=0.3 with [P]=0.16 millimolar 62 Figure 3-10 Initial Rate of DNA Crosslinking by HN-2 at 37oC. [Drug]/[P]=0.37 with [P]=0.16 millimolar, Trial 1 63 Figure 3-11 Initial Rate of DNA Crosslinking by HN-2 at 37oC. [Drug]/[P]=0.37 with [P]=0.16 millimolar, Trial 2 64 Figure 3-12 Summary of Dependence of Initial Cross-Link Rate on HN-2 Concentration at 37oC 65 Table 3-1 Summary of Initial Reaction Rates of PAM and HN2 67 Table 3-2 Pseudo First Order Rate Constants vu Initial Reaction Rates as a Function of Drug Concentration for PAM and HN2 68 Figure 3-13 HN2 Reaction Mechanism for Crosslinking DNA Via an AzMdinium Ion 71 Figure 3-14 A Possible Mechanism for PAM Crosslinking of DNA Via a Carbocation 72 Figure 4-1 Generalized Arrhenius plot 75 Figure 4-2 Initial Rate of DNA Crosslinking by PAM at 24.5oC.