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

Abstract

Because this article is a bit of history rather than ascientific paper, we will depart from the formality of ajournalarticle.Thisarticlehastwopurposes.Thefirstistogive credit to two deceased individuals, Albert Castro,M.D., and Howard Gratzner, Ph.D. (1). Castro was the keyperson who was able to produce a polyclonal antibodythat was specific for bromodeoxyuridine (BrdU). For 10years, Gratzner took the lead in converting an idea into afunctional assay that affected DNA analysis and led to thedevelopment of many other assays with similar method-ologies. The second is to inform young scientists that (a)inventions or creations have utility in fields other than thepurpose for which they were created; (b) it is possible todo useful work in an institution that certainly would nothave been classified as one of the best; and (c) the pres-ence of talented people who are willing to collaborate isinvaluable.Scientifically, Gratzner is best known for the develop-ment of the first monoclonal antibody (mAb) to BrdU andiododeoxyuridine. This work was the culmination of pre-vious efforts with Robert Leif to develop polyclonal anti-bodies, which detected BrdU with variable specificity.Collaborations with scientists at the Lawrence LivermoreLaboratory encouraged the development of a widely usedflow cytometric test for the simultaneous measurementsof DNA and BrdU. The use of mAbs to halogenated pyri-midines was a significant scientific advance and currentlyis an integral tool for the analysis by flow and digitalmicroscopy of the cell cycle.On a personal level, Gratzner’s enthusiasm for scienceand his intelligence, curiosity, loyal interactions with fel-low collaborators, and friendship are sorely missed by allwhoknewhim.Hewasgenuinelyawarmandnicepersonwho was liked by almost everyone with whom he came incontact (1).Castro was a jovial man who interacted well with hiscollaborators. He was generous and very knowledgeable.He ran an immunodiagnostics laboratory at the universi-ties of Oregon and Miami and was an expert in the pro-duction of antibodies in animals. His warm smile, gener-ous giving of his knowledge, common sense, enthusiasmfor scientific research, and ability to get the job done wereessential to this project.Because this is a story of events that happened approx-imately 30 years ago and the existing records are limited,the accuracy of this article is limited by our memory andthose of our colleagues who have kindly helped with thisproject. Of course, this is our version of the events.Cell division and DNA replication are fundamental tobiology and specifically to the biology of cancer. Thisarticledescribeshowasimpleprocedureforthedetectionand quantitation of DNA synthesis was developed. Theprecise determination of when S phase occurs in the cellcycle is of use to maximize the selective killing of tumorcells with cycle-specific chemotherapeutic drugs. Ofgreater significance, studies of the control of the cellcycle, including its detour into apoptosis, can provideextremely useful insights into the creation of new thera-peutic regimens.The original process to detect S-phase cells included theuse of tritiated thymidine with autoradiography to mea-sure the labeling index (percentage of S-phase cells)and/or the fraction of labeled mitotic cells. Before and atthe infancy of flow cytometry, it was believed that thequantitative DNA analysis of normal and neoplastic cellsmight provide an objective marker for the diagnosis ofneoplasia. These measurements were performed with amicroscope on Feulgen-stained cells. In a normal popula-tion, it was established that there were two predominantpeaks in the DNA distribution, with a ratio of 1:2 in DNAcontent, and that cells that were synthesizing DNA (Sphase) were scattered in between, with a relative DNAcontent between 1 and 2. The detection of tritiated thy-