Measuring DNA
Synthesis Rates
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with Stable Isotopes Although stable isotopes are not hazardous in vitro or in vivo, using them to measure DNA synthesis rates has not been attempted until recently.
T
he biochemical method of studying cell proliferation involves measuring the rate of DNA synthesis. To do this, some form of labeled nucleoside must be incorporated into ongoing DNA replication. Currently, three approaches exist: radionucleosides, halogenated pyrimidines, and stable isotopes. Radioactivity, in the form of [3H]thymidine, represents the standard approach in cellular systems but is rarely used in experimental animals and never in humans. Bromodeoxyuridine is the most widely used halogenated pyrimidine. The fluorescence it generates is useful, but it is toxic to cellular systems. Stable isotopes, however, are not hazardous in vitro or in vivo. Despite the wide use of DNA-labeling methods in experimental biology, the stable isotope approach had not even been attempted until recently. In this article, we discuss three existing methods using stable isotopes to measure DNA synthesis rates (1–3). Beyond the results of these three procedures, we provide perspectives on choosing labels, preferred precursor species, and optimum instrumentation. Finally, we discuss the current and future applications of these methods.
©2003 TERESE WINSLOW
Gavin E. Black Fred P. Abramson George Washington University School of Medicine and Health Sciences
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Theory
When selecting a labeled precursor, both the chemical naThe biochemistry of DNA synthesis is well described (4). DNA ture of that compound and its isotopic composition must be is a polymer of nucleotides comprised of deoxyribose, phosphate, carefully considered. These choices determine the level of DNA and purine or pyrimidine bases. Two distinct pathways lead to the enrichment, the amount of precursor necessary to attain that formation of these nucleotides: de novo synthesis and salvage level, and the selectivity of the labeling. The highest level of en(Figure 1). (The balance between de novo synthesis and salvage richment that can be obtained in the targeted DNA subcomis unclear and varies among different cell types.) Purine bases are ponent is directly dependent on the level of enrichment of the synthesized de novo on the sugar–phosphate moiety, forming precursor. That level of enrichment will not be reached unless guanine or adenine (Figure 2). The whole glycine molecule is the labeled species is a component of every route to DNA synincorporated along with two nitrogens from glutamine, one from thesis. Thus, glucose is the only precursor that might accomaspartate, and two carbons from N 10-formyltetrahydrofolate. plish a parallel labeling of product and precursor because it enDe novo synthesis of pyrimidines (Figure 3) begins by car- ters both de novo and salvage pathways. On the other hand, bamoyl phosphate binding to aspartate followed by the addi- labeled bases or nucleosides go directly into the salvage pathtion of deoxyribose and a phosphate from 5-phosphororibosyl- way and will contribute to a large enrichment only if salvage is 1-pyrophosphate and a nitrogen from glutamine to yield cytidine the predominant route in the system under investigation. A second consideration when choosing the label is how and thymidine. The salvage pathway reutilizes both the bases and nucleosides from nucleotide degradation. Any of the chem- much material it takes to produce the desired enrichment of its ical species in these two pathways can be modified to label DNA. precursor pool. The higher the concentration of an endogeThe most direct strategy incorporates labeled bases or nucleosides into newly synthesized strands of DNA via the salvage pathway. Other strategies use glycine, glucose, or water. In addition to incorporation into purines, the C2 position of glycine may be converted into either the C2 or C3 position of serine. The C3 position of serine Nucleotides is involved in one-carbon transfers through a To DNA From DNA tetrahydrofolate derivative, which, in turn, is part of the de novo thymidine biosynthesis (4). Thus, Nucleotidases PPi [1,2-13C2]glycine might not selectively label Nucleoside kinases purines without altering the natural abundance of the pyrimidines. Glucose is converted into dePhosphoribosyl oxyribose and labels all the deoxynucleotides. Nucleosides transferases Neese et al. discuss the metabolic sources of deuterium in each position of the deoxyribose ? moiety of a nucleotide using 2H2O as the source Phosphorylases PRPP of the label (6). They measured enrichment in their deoxyribose derivative at [M + 2], reflectNucleic bases ing the incorporation of two atoms of 2H. Many Glucose mechanisms may be available to generate deuterium substitution into the bases, but we are unaware of data that indicate what the labeling pattern might be. FIGURE 1. Salvage pathways of DNA synthesis.
Methodology A procedure to measure DNA synthesis with stable isotopes involves several steps: A labeled precursor is selected and administered in a way that allows incorporation into DNA, the DNA from cells is extracted and hydrolyzed to monomeric nucleosides or their degradation products, the products are separated chromatographically, and the enriched products are measured by MS. 58 A
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Nucleic bases (thymine, cytosine, guanine, or adenine) can be converted into the nucleotide monophosphates (thymidylate, cytidylate, guanylate, or adenylate) directly by phosphoribosyl transferases that use phosphoribosyl pyrophosphate (PRPP). The ribose in PRPP comes from glucose via glucose-6-phosphate. An alternative two-step process also occurs, where the bases are glycosylated (presumably also using some glucose-derived species) by processes that are not well described (represented by the question mark). These nucleosides (thymidine, cytidine, guanidine, and adenine) can then be phosphorylated. Thus, glucose, nucleic bases, nucleosides, and nucleotides can all be used as stable isotope-labeled tracers to measure DNA synthesis. These intermediates are shown in orange. The pale yellow arrows show movement toward DNA, and the red arrows show degradation pathways. (PPi is pyrophosphate.)
nous DNA precursor species, the greater the amount of labeled material required to reach a given level of enrichment. In this regard, glucose is far from ideal. The concentration of glucose in cell culture medium or plasma is high, up to 4 g/L for medium and 1 g/L for plasma. In contrast, the concentration of glycine in plasma or medium is a few percent of glucose. The concentrations of nucleosides are orders of magnitude lower, ≤1 µM. Thus, each milligram of glycine generates more enrichment of its pool than each milligram of glucose. A milligram of a nucleoside represents an even larger fraction of its pool. This consideration affects only the economics of an experimental approach, not its outcome (6). A third consideration regarding the tracer species is its selectivity. If isotope ratio MS (IRMS) is used to measure the isotopic content of DNA-derived nucleosides, differences in the baseline 13C/12C may result from differences in the subjects’ or specimens’ diets, which might create uncertainty. Experimental researchers must determine whether a change in the 13C/12C
ratio is due to diet or tracer. Thus, for the highest-sensitivity work, glycine or a nucleoside is preferred because at least one nucleoside will control for one’s diet and can serve as an internal IR baseline. Different considerations are involved in the selection of the isotope. The natural abundance of 2H is 0.015%, the lowest of the common biological tracers. The natural abundances are 0.37% for 15N and 1.1% for 13C. Deuterium is inexpensive, and more deuterated chemical species are commercially available than for any other stable isotope. 13C- and 15N-labeled compounds are generally several times more expensive than 2H-labeled ones and much less commercially available. Deuterium has problems with kinetic isotope effects and exchange reactions, which are not important considerations for isotopes of carbon or nitrogen. Finally, the absolute numbers of atoms in a target molecule must be considered. Generally, there are many carbons and hydrogens in a given monomeric biomolecule but rarely more than one or two nitrogens. The analyst
NH2
Glycine Glutamine CO2 Formyl Aspartate
O N
HN
Glucose Hexokinase
H 2N
PO42–
N H2C
N O
Carbamoyl phosphate
Aspartate Glutamine Carbamoyl phosphate N 5, N 10-Methylene tetrahydrofolate
N
+ Aspartate
O PO42–
N O
H2C
Glucose-6phosphate Orotate
OH
NH2
Deoxyguanylate
N
N
PRPP
N PO42–
H2C
CH 3
N
Deoxycytidine PRPP O
N O
Orotidylate
Deoxyadenylate
FIGURE 2. Pathways for the de novo biosynthesis of purine nucleotides. The synthesis starts with PRPP, which has been formed from glucose. The multistep process begins at the ribose of PRPP as the purine ring is formed from a variety of precursors. The central product is inosinate. From there, atoms are added or replaced when generating guanylate or adenylate. Before becoming DNA, the ribose is reduced to deoxyribose.
PO42–
N O
H2C
OH
OH
Inosinate
O
OH
+
Uridylate
Thymidine
FIGURE 3. Pathways for the de novo biosynthesis of pyrimidine nucleotides. The synthesis starts when carbamoyl phosphate and aspartate combine to form orotate, which is then phosphoribosylated by PRPP to form the phosphoribonucleotide orotidylate. Uridylate is the next intermediate before a series of steps that create deoxycytidine and thymidine. There are fewer precursors that can be used as isotopic tracers in pyrimidine biosynthesis than there are for purine biosynthesis.
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Given this perspective, the best currently available scheme for stable isotope label-based measurements of DNA synthesis rates is GC/COM/ IRMS. The only drawback to using GC rather than HPLC is the need for derivatizations that add carbon and dilute and skew the IR. For example, making the pentaacetate derivative of glucose adds 10 carbons to the 6 from glucose and lowers the apparent enrichment by more than half. Another problem arises when derivatizing agents containing Instrumentation Conventional mass spectrometers are capable of determining silicon are used. Each atom of silicon contributes 5.1% to [M + enrichment in the range 100–0.2 atom percent excess (APE) 1] and 3.4% to [M + 2]. The presence of a complex isotopic enwith a precision of 0.2% (8). [APE is defined as 100(IR enriched – velope limits the detection of enrichment. To measure IRs, we IR natural)/(IR enriched – IR natural + 1) for the two masses being recommend using the derivatizing agents with the least contrimonitored and denotes the composition of a species in terms of bution to the isotope envelope, at least if the increment of mass the percentage content of the minor isotope compared with the due to the heavy atoms is only 1 or 2. In contrast, measuring natural abundance. APE is one of several terms that can be used combustion products such as CO2 avoids concerns about the to describe isotopic enrichment data (7).] On the other hand, complexity of the original isotope envelope and the presence of IRMS can measure approximately 2 orders of magnitude lower other elements besides the one representing the tracer. enrichment than conventional MS. Commercial instruments With conventional MS of intact analytes, measuring the IR combining GC, a combustion unit, and IRMS (GC/COM/ is achieved by integrating signals at [M + n]+/[M]+, where n is the increment in mass achieved by the isotopic substitution. When the target species is known, as is the case with nucleosides from DNA, Using IRMS allows much lower this procedure is straightforward. For example, the enrichment of the trimethylsilyl (TMS) derivatives of detection limits than conventional MS, deoxyguanosine (dG) labeled by [6,6-2H2]glucose was measured at perhaps 2 orders of magnitude. m/z 555 and 557, and the similar preparation of deoxyadenosine (dA) at m/z 467 and 469 (1). In contrast, reduction of these analytes would generate H2 and HD at m/z 2 and 3. The chromatoIRMS, [8]) are capable of measuring the IR of the CO2 pro- graphic retention time provides the only qualitative informaduced by the combustion of analytes to better than 0.001 APE. tion on species if the structure-destroying steps of atomization HPLC/COM/IRMS units are not commercially available, and reduction are used. The two great advantages of the atalthough two have been described. Caimi and Brenna used a omization scheme are that the baseline IR for HD at m/z 3 is moving wire to send HPLC effluent through a CuO combus- only 0.015%, which provides high abundance sensitivity, and tor that fed into an IRMS instrument (9). Standard deviations that the IRs for all analytes are continuously monitored, giving of
