A short history of the initial application of anti–5‐BrdU to the detection and measurement of S phase

Because this article is a bit of history rather than a scientific paper, we will depart from the formality of a journal article. This article has two purposes. The first is to give credit to two deceased individuals, Albert Castro, M.D., and Howard Gratzner, Ph.D. (1). Castro was the key person who was able to produce a polyclonal antibody that was specific for bromodeoxyuridine (BrdU). For 10 years, Gratzner took the lead in converting an idea into a functional assay that affected DNA analysis and led to the development of many other assays with similar methodologies. The second is to inform young scientists that (a) inventions or creations have utility in fields other than the purpose for which they were created; (b) it is possible to do useful work in an institution that certainly would not have been classified as one of the best; and (c) the presence of talented people who are willing to collaborate is invaluable. Scientifically, Gratzner is best known for the development of the first monoclonal antibody (mAb) to BrdU and iododeoxyuridine. This work was the culmination of previous efforts with Robert Leif to develop polyclonal antibodies, which detected BrdU with variable specificity. Collaborations with scientists at the Lawrence Livermore Laboratory encouraged the development of a widely used flow cytometric test for the simultaneous measurements of DNA and BrdU. The use of mAbs to halogenated pyrimidines was a significant scientific advance and currently is an integral tool for the analysis by flow and digital microscopy of the cell cycle. On a personal level, Gratzner's enthusiasm for science and his intelligence, curiosity, loyal interactions with fellow collaborators, and friendship are sorely missed by all who knew him. He was genuinely a warm and nice person who was liked by almost everyone with whom he came in contact (1). Castro was a jovial man who interacted well with his collaborators. He was generous and very knowledgeable. He ran an immunodiagnostics laboratory at the universities of Oregon and Miami and was an expert in the production of antibodies in animals. His warm smile, generous giving of his knowledge, common sense, enthusiasm for scientific research, and ability to get the job done were essential to this project. Because this is a story of events that happened approximately 30 years ago and the existing records are limited, the accuracy of this article is limited by our memory and those of our colleagues who have kindly helped with this project. Of course, this is our version of the events. Cell division and DNA replication are fundamental to biology and specifically to the biology of cancer. This article describes how a simple procedure for the detection and quantitation of DNA synthesis was developed. The precise determination of when S phase occurs in the cell cycle is of use to maximize the selective killing of tumor cells with cycle-specific chemotherapeutic drugs. Of greater significance, studies of the control of the cell cycle, including its detour into apoptosis, can provide extremely useful insights into the creation of new therapeutic regimens. The original process to detect S-phase cells included the use of tritiated thymidine with autoradiography to measure the labeling index (percentage of S-phase cells) and/or the fraction of labeled mitotic cells. Before and at the infancy of flow cytometry, it was believed that the quantitative DNA analysis of normal and neoplastic cells might provide an objective marker for the diagnosis of neoplasia. These measurements were performed with a microscope on Feulgen-stained cells. In a normal population, it was established that there were two predominant peaks in the DNA distribution, with a ratio of 1:2 in DNA content, and that cells that were synthesizing DNA (S phase) were scattered in between, with a relative DNA content between 1 and 2. The detection of tritiated thymidine incorporation by autoradiography was the technique for marking cells in S phase. The immunochemical measurement of S phase greatly facilitated cell cycle analysis of samples that were a mixture of normal and aneuploid cells. This communication describes the history of the early development of this technique. Early flow cytometers had the capacity to detect one fluorescence parameter in addition to light scatter parameters from cells or nuclei. Van Dilla et al. (2) initially used the fluorescent Feulgen reaction to quantitate DNA and observed the G1 and G2 peaks of the cell cycle. Subsequently, Crissman et al. (3) and Krishan (4) replaced this acid-based technique by the use of DNA-binding fluorochromes. The capacity of flow fluorometry to precisely measure the DNA content of a statistically significant number of cells greatly improved cell cycle analyses. However, flow cytometers are unsuited for conventional autoradiographic measurements including that of tritiated thymidine incorporation. This article describes a fluorescence method, the use of a fluorescent antibody, to detect the incorporation of BrdU into DNA. There are three major technologic requirements for the clinical flow cytometric measurement of 5-BrdU incorporation into cellular DNA: (a) 5-BrdU must be made available to and incorporated by the cells; (b) single-cell measurements by flow cytometry have always required producing a monodisperse cell suspension; and (c) there has to be a means to detect the incorporation. The incorporation of 5-BrdU can be accomplished by direct injection of the labeled nucleotide into the patient, labeling of tissue fragments or slices, or adding 5-BrdU to single cells in a culture medium. Direct injection of a labeled nucleotide into humans is potentially harmful and requires a large amount of an expensive compound. Because tissue fragments or slices no longer have molecules delivered by the circulatory system, these molecules, including the labeled nucleotide, must be delivered by diffusion. The limitations on diffusion imposed by the tissue architecture will result in spatial differences in concentration. Another possible solution is to dissociate the tissue slices or fragments into single cells before exposure to the labeled nucleotide. In any event, after in vivo and before or after in vitro incorporation, monodisperse cell preparations have to be created. The simplest solution is to produce nuclei; unfortunately, the loss of the cytoplasm very often precludes the ability to connect the individual DNA measurements with a specific type of cell. Dissociation of tissue slices or fragments was and remains problematic. Although in hindsight it seems logical to detect S phase by the fluorescence of antibody reacting with BrdU that had been incorporated into cells, it should be emphasized that in the mid-1970s this was far from guaranteed to work. In the mid-1970s there was limited application of antiserum against biological molecules, and the creation of mAbs had not yet occurred. The technical difficulties of reproducibly producing a polyclonal antibody specific for 5-BrdU were significant. Although the introduction of this article has provided a rational and orderly view of the development of anti–5-BrdU, this was not the case. The application of anti–5-BrdU for the detection and measurement of S phase was the result of necessity, not of foresight or wisdom. In essence, one invention by Leif, centrifugal cytology, forced his invention of the use of anti–5-BrdU for measuring S phase. Centrifugal cytology is a process that centrifuges cells onto a slide and then fixes the wet cells under the action of centrifugal force. This process is based on G. N. Papanicolaou's great technical contribution to cytology, wet fixation (5). The surface tension forces produced by air drying from water are sufficient to disrupt and severely distort the morphology of cells, in particular active cells. Because linear gradient buoyant density centrifugation of human erythrocytes (6), guinea pig bone marrow (7), and other cells (8, 9) had indicated that buoyant density is related to cell age, it became of interest to perform tritiated thymidine labeling (10). Conventional liquid emulsion technique required air drying from the emulsion, which distorted the cellular morphology. Marilyn Cayer, one of Leif's longest term collaborators, provided a solution to this problem. Fortunately, she was and is a very talented electron microscopist. She embedded cells that were bound to the microscopic slide, sectioned them in the direction parallel to the microscopic slide surface, and then covered the sections with liquid emulsion. This resulted in a preparation that allowed reliable counting of silver grains. Unfortunately, the technique was somewhat arduous and time consuming and could be performed only by a few superbly talented individuals. Frankly, Leif certainly was not one of them. He therefore started a dialog with Gratzner concerning the use of 5-BrdU. Gratzner was the most knowledgeable scientist concerning molecular biology at the Papanicolaou Cancer Research Institute (PCRI) and was willing to spend time the problem. Leif's as a in laboratory at he had known that 5-BrdU could be incorporated into DNA. The incorporation of 5-BrdU by cells the buoyant density of DNA and the by density gradient centrifugation of DNA from DNA. The anti–5-BrdU story one in Leif was at a on that all of his previous were the not an antibody against 5-BrdU and use it to detect the incorporated Leif Gratzner on the and his Gratzner it made For Gratzner and Leif, this a significant problem. Leif had made an and it is not known Gratzner Leif that Gratzner had done with a Gratzner and Leif to Castro, who had been by the of Miami to an For Castro, this was not a Castro, in addition to an expert on antibody was extremely and his Castro was a very large He had been in and he not only with his and became the of the project. Gratzner and Leif that and had made an antibody against 5-BrdU Castro had a polyclonal antibody, and Gratzner, with from one of Leif's it initially by diffusion and Gratzner and Leif were very and Castro was not that they were able to studies that a with and not with or the The of the of the by the presence of 5-BrdU in the provided The DNA was than the original in in this was from colleagues at the of Miami of who in DNA Leif that who had done studies on halogenated or one of his colleagues in the of at the of Miami was the one who the of the incorporation of 5-BrdU into DNA by adding to thymidine by Gratzner and Leif have been and have been on Leif's This describes the development of an for DNA synthesis in single cells, antibody specific for The technique of for the measurement of cell analysis in a of The technique to be an to autoradiography and has for flow quantitation of DNA in single cells or nuclei. had been were to the Cancer Institute and the of and always a in scientific including those at the the of the the on which was the of the of it was essential for all the there to including the to the in it was to made scientific to by sections and the scientific Unfortunately, Gratzner's and these have to do with the capacity to do scientific in the of the Gratzner not into the of In Gratzner was in of his was and scientific for of of the was Gratzner to his and at the Gratzner was the for the 5-BrdU which was a significant of the Gratzner included from Leif's Marilyn Cayer, the laboratory the and Leif's as included of and the of the content of the and to to the second was to the Cancer of the to the development of this 5-BrdU assay to S-phase cells by flow or The application in this was to the to use cell cycle to the use of This included the for an expensive fluorescence microscope to the 5-BrdU This microscope was essential for this and other at the Robert was a on the had been a in the laboratory at of at Leif had a had from a at the Institute in and was for his first at the on from Leif's and who was on cell The the with Gratzner as the and and Leif as Although the was extremely with the of the they were with the of Gratzner's the The believed that Gratzner not have the to lead this project. to the to the only was the and Gratzner was the The was not to a with the and to the of his two Although this is an the can be to the and the of the can the Gratzner was a who could not with the to people that he could get the job He was a and including science and individuals. In Gratzner's of was a credit to his scientific of the was based on work by Leif's on the of which in hindsight was than In had a and a for 30 years and he took on the of a for cells with the of measuring with a flow and a However, his was as he three other on from his and including the Leif helped to the best of his ability to this 5-BrdU to the However, Leif had of his including a flow that had the capacity to do simultaneous analysis in addition to work on his on cell In Gratzner was the person who performed the and his to the and ran the to He the and on this of antibody detection of S-phase cells) for many The scientific based on this work at the provided the scientific with a simple reliable to cell cycle and are to the it is and the ability to do the best possible job with that are essential to the of the project. the with the polyclonal antibody to 5-BrdU was in the of the on which was the of the was made of the labeling index by anti–5-BrdU and The a The with incorporation of as by liquid counting with the measurements was at best and Leif's in of cellular Gratzner the a of 5-BrdU labeled cells was an Because Gratzner was far knowledgeable than Leif tissue Gratzner the incorporation Gratzner was for the had Leif's and was with Gratzner 5-BrdU He performed many useful 5-BrdU and these a of his of He to a Ph.D. and an He is a clinical The was an article produced at the of of in which Leif and his many of the and for and other cytology including one by Leif in a by and the early history of that Leif was under the that flow cytometry was the of for this However, work an to dissociate cells into the single-cell for flow cytometric Leif, the of his was not a first on these However, the article on the and the work of his and he the of the Leif took the of first The of the of the use of DNA synthesis as one of the of and how the produced by the replication of the in the samples could be by the use of The and to counting of silver with a microscope could be replaced by an flow cytometric Gratzner and Leif on the possible use of antibodies against the DNA that in S phase and the of work with first the of the of the tritiated thymidine autoradiography and then an of a fluorescent that had been for 30 with BrdU. application of antibody has been for the analysis of cell had to in the of the would have to be to an of a liquid for 30 before Although this is not to the for it was for and of which could be by centrifugal Because this a molecular to the of cells, it included a of the use of as and the use of centrifugal cytology for the detection of The on that helped Gratzner was his work on the was to the of Miami and The had to its by scientific between and the Cancer at the of of the at the including Leif with this and believed that it would and at the of Miami and the with the which could result in Because Leif had any to Gratzner, the used the of as the to has its it has the of Fortunately, had started the Institute for Cell at the of Miami with generous She Gratzner as a of the of which allowed to the 5-BrdU Gratzner that he a at the of in to his and took a on at the in The greater of events a in his to to the early it that the of the on would provide a of for The was a very time for scientific in years, almost of were would the be any to Gratzner that at a was very and would be not a major in his scientific However, as and on in this of this was not the and was a in his after Gratzner the Gratzner and Leif the work to produce the the use of anti–5-BrdU for had that S-phase technique had to be with a quantitative measurement of DNA. Leif's first in he was well with the of by reaction of a with an he a Feulgen reaction in which the to the pyrimidines would be used to he the would the from the were in and in a solution of in for 1 normal and for 30 to this which is a and which in of and antibody to the incorporated this the Unfortunately, the of the Gratzner used was of For one Krishan kindly use his and we that a DNA The cell preparation procedure was it was to the DNA to DNA that could with the detection of 5-BrdU has been improved by based on the anti–5-BrdU which not of DNA. These have been in the of The technique of labeling by by laboratory was it the of The direct of anti–5-BrdU to the 5-BrdU in has been by et al. technique for the measurement of cell by an has been The of to be detected in DNA. the human cell used in this of 30 can be This the of of this technique is the between the amount of incorporated into as by and the fluorescence produced by specific antibody to This that the amount of DNA in individual cells can be for analysis of on DNA or as well as other of an to the autoradiographic procedure as to the conventional DNA method, is that it of cells to be of DNA content as they the cell cycle a second as DNA content or cell were available it would be possible to the of cells specific for the measurement of cell cycle parameters The flow cytometry control cells which was to the light scatter Fortunately, the contribution of the creation of a to mAbs by and this problem. that Leif's for the creation of the for cells not Leif had most of the previous in on including development of the first that ran on a this was sufficient only to produce a He not have a reliable to use for flow cytometric kindly Leif's at the the in the was by individuals, the of the Because the was very with with the including the of and Leif's with the Miami Cancer after a Leif was In the to The was and replaced by a with the of The of Miami scientists with who was for the 10 previous years, in early He Miami in early and to work for the as a scientist a flow cytometry The had the great of a of extremely and N. Papanicolaou and who made very significant to centrifugal cytology and the of Leif's work on preparation Gratzner, Leif, and to with scientists as Marilyn Cayer, and Leif to his and to biological studies with that direct and The analysis was an of this work. Frankly, the of clinical is great that it was of Leif to that he could the of a laboratory at a Another based on the early was and at by who was one of Leif's at the the by and it was that a against 5-BrdU should be created. Gratzner was at the Institute for Cell on the of Miami and by Gratzner a who had in cell which is an essential of the technique by and for mAbs to specific was to produce a against 5-BrdU with greater and than that of the polyclonal were with to the procedure of and of the of the of by for a with the of In with the of and cells were from an reaction and with cells. The cells, were and in and the culture were for antibody by The very first by in assay was and to a the anti–5-BrdU was to have for and a quantitative reaction with the was at microscopic preparations of cells with BrdU and then with anti–5-BrdU and antibody the of the application of this to cytology and to the measurement of the of DNA by flow cytometry These were by Gratzner and in studies of DNA replication by flow cytometric analysis and fluorescence microscopy of of early the interest and development of this of the work on anti–5-BrdU was made in Gratzner's laboratory by and a against with the under The objective of these as yet was to use this as a in determination and of the of in as a of and DNA the early of Leif's at he that its under the that the Institute for Cell to Gratzner's He was in the Research and his new were a when Leif the and the of Gratzner's Leif that it would be a great for flow cytometers and that he had a which of course, had to Leif, always for that is the of and to his This is the Leif any work on anti–5-BrdU at Subsequently, the of Miami had the to Gratzner's against 5-BrdU and its and to and use the are all by the of that its contribution to in the cell cycle and cell and its application for of the S phase. The ability to for a not only sufficient foresight with In this with anti–5-BrdU the of and are that and the of Miami had the foresight to develop the Unfortunately, at the the of had from the of the the 5-BrdU which the had the between and the Institute for Cell in Gratzner was The of the to the of Miami to the of Gratzner had been a could not be used to Gratzner he was a of the of and Gratzner first to and then to Livermore he with which anti–5-BrdU and the of this to the cell cycle were able to measure DNA content and 5-BrdU antibody a at Gratzner to he with the and cell at and with scientist at the and the cell cycle with the 5-BrdU Because of a Castro had been to an of the of Miami and there his Leif, after to and has to with The for S phase and apoptosis, which are based on in laboratory have been to use a labeled with anti–5-BrdU the became a scientist at the in Research and is a flow cytometry and microscopy Cell cycle analysis is a key of his was not a to this anti–5-BrdU project. However, the of S-phase content and would be useful for the to for the The use of to the cell cycle would no longer be and cells in S phase could be with The flow cytometric were of and the antibody could with single and the could be as a flow cytometers that in the and early were of fluorescence The scientists in Miami initially at the and then at the Institute for Cell on this project. delivered a that was of by into a that the the cell cycle was The use of anti–5-BrdU is a procedure that has facilitated cell cycle studies and provided a for the use of for the of in cell nuclei. The these studies was to provide a technique to cancer. to the new and of flow cytometry helped provide this tool to other scientists to the cell cycle in of microscopy and flow This useful work was accomplished by a of with There are to be from this anti–5-BrdU do science often the and of The not have sufficient to this or the or an with the to the of this work. In the to a on this has to be that our was in to the of our in to our to produce a useful for the and scientific and most to the with should be to the and which had the to the utility of the This work would not have been the of new technique to cells in the S phase was a scientist had to a with his Conventional autoradiography required air drying from which was with the used by the in a that this bit of history has that it is not for everyone to all the and new In a scientific it is for to a idea and work on initially we have in by as a we were all able to the development of this 5-BrdU the for of and with this Marilyn Cayer, Leif, and