The cytoplasmic enzyme ribonucleotide reductase is essential for DNA synthesis, and its activity is strongly correlated with cellular proliferation. This paper describes a flow cytometric technique for the simultaneous measurement of DNA content and the M1 subunit of ribonucleotide reductase. Data are presented for cycling cultured human leukemic lymphoblasts in which M1 is constitutively expressed, and peripheral blood lymphocytes in which it is only detectable with certainty after mitogen stimulation.
The choice of fixation procedure strongly influenced the amount of M1 subunit de-tected. Paraformaldehyde (PF) at concentrations of 2% (wlv in PBS) or greater provided optimal results. Fixation at 3'7ยฐC was significantly more effective in preserving M1 than fixation at room temperature or 4ยฐC. These variables are shown to have affected cytoplasmic retention during postfixation processing. Their relevance to the flow cytometric measurement of other intracellular components by this procedure are discussed.
Key terms: Cell cycle, cytoplasmic proteins, paraformaldehyde, propidium iodide Ribonucleotide reductase catalyzes the conversion of ribonucleoside diphosphates to their corresponding deoxyribonucleotides. This is the first unique, rate-limiting step in DNA synthesis. Biochemical studies have shown that the activity of this enzyme is high in proliferating tissues and suggest that catalysis only takes place during S phase (16).
Recently, monoclonal antibodies have been raised against the ribonucleotide reductase M1 subunit (a polypeptide of Mr 80,000 which binds allosteric effectors) and have been used in a sensitive biochemical immunoassay and in histochemical studies (3,5,6). The latter work showed that the M1 subunit is cytoplasmic and detectable only in proliferating tissues. We therefore sought to develop a flow cytometric technique to quantify ribonucleotide reductase M1 subunit levels by immunofluorescence, correlate these measurements simultaneously with DNA content, and directly examine cell cycle effects on enzyme content.
Flow cytometric techniques for surface immunofluorescence studies are well established, but measurement of intracellular antigens is less easy to achieve because of the need to render the cell permeable to antibody without denaturing or dispersing the target molecule. An increasing number of papers have described flow cytometric analyses of intranuclear antigens (reviewed