Howard G. Gratzner

Cell-Cycle Analysis Using A Monoclonal Antibody to Brdurd

The flow cytometric measurement of DNA distributions of cells has many applications in biomedical research. Phase fractions estimated (calculated) from such distributions are used to study the growth characteristics of various types of cells, particularly when the cells have been exposed to perturbing agents such as chemotherapeutic drugs. For more than 10 years many methods for resolving DNA distributions into the three cell subpopulations (G1, S and G2, + M) have been reported in the literature. A new method of analysis utilizing a monoclonal antibody to bromodeoxyuridine (BrdUrd) has been developed (Gratzner, 1982; Dolbeare et al., 1983) which makes it possible in most cases to accurately determine phase fractions without resorting to mathematical models. the procedure involves the incorporation of BrdUrd by growing (DNA synthesizing) S phase cells, labelling the BrdUrd with a fluorescent monoclonal antibody, and the bivariate measurement of the antibody and of total DNA content, the latter through propidium‐iodide staining. the resulting bivariate distributions clearly and simply resolve the three subpopulations. This paper describes the method and illustrates its use in the analysis of various fractions of elutriated exponentially growing Chinese hamster ovary (CHO) cells.

The interaction of bisulfite and its free radical analog-nitroxyldisulfonate with periodic acid-oxidized lymphocytes and their effect on blastogenesis

Nitroxyldisulfonate [Fremys salt; (KSO3)2NO.] and bisulfite (NaHSO3) have abolished periodic acid (H5IO6)-induced blastogenesis of human peripheral blood lymphocytes (HPBL), but only inhibited the blastogenic response of H5IO6-oxidized rat and mouse lymphocytes, as determined by the rates of nucleic acids synthesis, BrdUrd incorporation and by cell numbers in S + G2 + M phases of the cell cycle. The viability of the intact human, rat and mouse lymphocytes remained essentially unimpaired by 30 min pulses of 1 mM Fremys salt or bisulfite. The marked inhibition of periodic acid-induced blastogenesis, exerted by Fremys salt and by bisulfite, was attributed to the effect of the corresponding carbonyl addition derivatives formed in situ of the oxidized cell membranes. Consequently, it is concluded that Fremys salt like bisulfite possibly forms addition derivatives with membrane carbonyls of viable target cells.

The Assessment of the Chemotherapeutic Effects of Vinca Alkaloids by Immunofluorescence and Flow Cytometry

The effects of the vinca alkaloids on the rates of DNA synthesis in the human pancreatic carcinoma line, MIA Pa Ca-2 have been studied by a new technique for measuring cell kinetics and DNA synthesis by flow cytometry and immunofluorescence. The method employs a monoclonal antibody that is highly specific for bromodeoxyuridine or iododeoxyuridine. The drugs vincristine and vindesine do not appear to have a direct effect on DNA synthesis rate across S phase, whereas DHAD, a compound that has been found previously to affect DNA synthesis, does appear by this technique to inhibit DNA synthesis at specific segments of S phase. That vincristine does not block cells in S phase by inhibiting DNA synthesis is borne out of the observation that cells blocked in S or G2 + M can still incorporate BrdUrd at a high rate in these phases of the cell cycle.

Application of Antibodies to 5-Bromodeoxyuridine for the Detection of Cells of Rare Genotype

A current objective of research in the area of environmental mutagenesis is the development of automated methods of assessing in vivo and in vitro genetic damage.(1,2) The primary purpose of automating mutagen detection is to increase the speed and statistical reliability that is afforded by the instrumentation being recruited to this task, flow or image cytometry. In addition, this approach would eliminate the human bias and error characteristic of manual methods. In the case of in vivo assessment of mutagenesis, a method would be available to measure genetic variation in cells that either do not clone or clone at extremely low efficiencies.