Proc. Nati. Acad. Sci. USA Vol. 74, No. 7, pp. 2889-2893, July 1977 Cell Biology Control of growth by picolinic acid: Differential response of normal and transformed cells (cell synchrony/chelating agents/NAD+/pyridine derivatives/viral transformation) J. A. FERNANDEZ-POL*t, VINCENT H. BONO, JR.J, AND GEORGE S. JOHNSON* * Laboratory of Molecular Biology and * Laboratory of Medicinal Chemistry and Biology, National Cancer Institute, Bethesda, Maryland 20014 Communicated by Nathan 0. Kaplan, April 14, 1977 ABSTRACT Picolinic acid reversibly inhibits the growth under 95% air/5% CO2, humidified atmosphere, at 37°. The of cultured cells. Fourteen other pyridine derivatives were in- cell lines used are listed in Table 1. Unless otherwise indicated, effective or toxic. Untransformed normal rat kidney (NRK) cells cells were planted at 1.5 X 105 cells per dish. Twenty-four hours are reversibly arrested in the G1 stage of the growth cycle as later, media were replaced by new media with or shown by cell counts, mitotic index, [3H]thymidine incorpora- (treated) tion, and flow microfluorometry. Flow microfluorometry was without (control) picolinic acid. All cells lines were treated with used to monitor the effects of picolinic acid on numerous other picolinic acid at 1.5,2,2.5,3, and 4 mM and analyzed for 4 days cell lines. Normal cells are blocked in GI, whereas transformed with media change every other day. cells show responses that are dependent upon the transforming Cells were analyzed for DNA content by flow microfluo- virus and independent of species or origin of the cell line. Kir- rometry (FMF) after trypsinization and suspension in pro- sten sarcoma virus-transformed cells are blocked in GI. Simian virus 40-transformed cells progress to a G2 block. Cells trans- pidium iodide solution (0.05 mg/ml in 0.1% trisodium citrate) formed by polyoma or Harvey sarcoma virus with Moloney virus at 2 X 106 cells per ml (3). The frequency distribution of fluo- coat have flow microfluorometry profiles that indicate blocks rescent emission per cell (proportional to DNA content) was in both G1 and G2. Cells transformed with Moloney sarcoma measured in 105 cells using a cytofluorograph (Biophysics virus are not blocked in a specific phase of the cell cycle. Systems, Inc., Baldwin Place, NY), and analyzed with a mul- Picolinic acid does not change the levels of NAD+ plus NADH; however, the growth inhibition by picolinic acidis partially tichannel analyzer (Tracor Northern, Inc., Middleton, WI). overcome by nicotinamide. These results suggest that picolinic NAD+ plus NADH was assayed as previously described (4). acid interacts with a specific growth control mechanism that Recovery of a known amount of NAD+ was greater than 95%; may involve NAD+ and that this control mechanism is altered no reactive material was detected in medium, and medium by different transforming viruses in different manners. containing 3 mM picolinic acid did not interfere with the assay. Cells were counted with a Coulter counter. Thymidine incor- Picolinic acid is a metal-chelating agent (1) and is also poration was measured (5) with 0.05 /ACi of [3H]thymidine per structurally related to nicotinic acid, a precursor in the bio- dish. synthesis of NAD+. On the basis of theoretical considerations on the role of metal ions and NAD+ in growth control (J. A. RESULTS Fernandez-Pol, unpublished), picolinic acid was tested and found to be a useful agent to study cell growth regulation. Effect of Picolinic Acid on Growth of NRK Cells. The Picolinic acid is of further interest because it is a biological growth of normal rat kidney (NRK) cells was inhibited by component in at least some cells and could participate in a picolinic acid in a dose-dependent manner (Fig. 1); the inhi- physiological growth regulatory mechanism. bition was reversible within 24 hr after picolinic acid removal. Here we report on the effects of picolinic acid on the growth These results, which were reproduced in four separate exper- of cultured cells. The agent is an inhibitor of cell growth and iments, suggested a possible block at a specific point in the cell reversibly arrests cells at a specific point in the cell cycle. A cycle. To test for such a block, NRK cells were exposed to 3 mM preliminary report on this work has been published (2). picolinic acid for 36 hr. At this time essentially no mitotic cells were observed. A wave of DNA synthesis began 11 hr after MATERIALS AND METHODS removal of picolinic acid (Fig. 2), followed by a burst of mitosis Materials. Nicotinamide, nicotinic acid, and a doubling in cell number from 16 to 22 hr. In this partic- picolinic acid, ular experiment 5-6 hr were for the cells to double NAD+, and 2,6-pyridine dicarboxylic acid were obtained from required in Sigma; picolinamide, from Matheson, Coleman, and Bell; iso- number after the first mitotic cells were observed. In other nicotinamide, isonicotinic acid, isonicotinic acid hydrazide, experiments this process was even more rapid, with a complete 3-aminomethylpyridine, 2,4-lutidine, 2-aminomethylpyridine, doubling within 2-3 hr. To quantitate and further verify the 2,3-pyridinedicarboxylic acid, and 3,5-pyridinedicarboxylic G1 block, FMF analyses were done (Fig. 3). The results clearly show that treated cells were in acid, from Aldrich. 3-Mercaptopicolinic acid was provided by predominately G1. Furthermore, H. Saunders, Smith, Kline and French, Philadelphia, PA, and when picolinic acid was removed, the number of cells in S and G2 increased and, Ca2+ ionophore A-23187 by R. J. Hosley, Eli Lilly Co., India- following the first wave of mitosis, essentially napolis, IN. all of the cells were in G1. Thus, NRK cells were arrested in G1 Methods. Cells were grown in Dulbecco-Vogt modified by picolinic acid and they proceeded through the cell cycle after its removal. Eagle's medium containing 10% (vol/vol) calf serum (Colorado Serum Co.) in 20-cm2 tissue culture dishes (Falcon Plastics) Effects of Picolinic Acid on Transfonned NRK Cells. The effects of picolinic acid were tested on three different trans- Abbreviations: NRK, normal rat kidney; FMF, flow microfluorometry; formed cell lines under conditions that arrested normal NRK SV40, simian virus 40. cells in G1. Cells transformed by different viruses responded t Present address: Department of Nuclear Medicine, Veterans Ad- differently. In all three cell lines there was no further increase ministration Hospital, St. Louis University, St. Louis, MO 63125. in cell number after 40 hr, but FMF analysis showed that the 2889 Downloaded by guest on September 28, 2021 2890 Cell Biology: Fernandez-Pol et al. Proc. Natl. Acad. Sci. USA 74 (1977) 1x Table 1. Cell lines investigated Transforming Type of Time, Dose, Cell lines virus block hr mM 4 X BALB 3T3 ClC/3 None Gi 48 3 BALB 3T3 C131-7 None G, 52-72 3-4 NRK Cl5W None G, 48 3 WI-38 ClA-17 None Gi 72 3-3.5 BHK-21 None G, + G2 48 2.5 KA31C01 BALB 3T3 KSV G, 43 2.5-3 K-NRK C132 KSV Gi 42-48 3 SV-BALB 3T3 C1SVT2 SV40 G2 48-72 2.5-3 1x SV-BALB 3T3 SV40 G2 48 2.5-3 ClSVTb3 SV-NRK P8 C12T7 SV40 G2 72 3 SV-WI-38 ClVA-13 SV40 G2 96-120 2.5-3 HSV/Mol-BALB 3T3 HSV with G1 + G2 72 2.5 4 X MSV coat A-PicolnicPioliicaidacidmedium-Normal HSV/Mol-NRK HSV with G1 + G2 72 2.5 .MSV coat PY-3T3 Clii Polyoma G, + G2 68 2 SV-PY-11 3T3 SV40 and G, + G2 68 2 polyoma 0 24 48 72 Time, hr MSV-BALB 3T3 C131 MSV Random 43-72 2.5 MSV-BALB 3T3 C185 MSV Random 48-72 2.5 FIG. 1. Dose dependence and reversibility of growth inhibition MSV-NRK MSV Random 43-72 2.5-4 in NRK cells treated with picolinic acid. Cells were planted at 5 X 104 cells per dish and 66 hr later the medium was removed and new media KSV, Kirsten sarcoma virus; HSV, Harvey sarcoma virus; MSV, containing picolinic acid at the indicated concentrations were added. Moloney sarcoma virus. BALB cells are mouse; WI-38 cells are human; Twenty-four hours later the media were removed, the cultures were BHK cells are hamster. rinsed once with fresh medium, and medium without picolinic acid was added. Cell counts were determined at the indicated times; each point is the average of duplicate measurements from two cultures. (Table 1). The latter point is clearly illustrated with the SV40-transformed cell lines. Four different lines progressed point of arrest in the cell cycle was different. K-NRK cells were through the cell cycle into G2 (Table 1). An example of this arrested in G1 (Fig. 4B). This stage of arrest was similar to that progression is shown in Fig. 6. SV40-transformed WI-38 cells observed with the parent NRK cells; however, it has not been (CIVA-13) after 53 hr began to accumulate in S and G2+M with established if the two cell lines were blocked at identical points a concomitant decrease in G,. The cells then further progressed in G1. Under these conditions simian virus 40 (SV40)-trans- into G2, and by 96 hr, most of the cells were in G2. formed NRK cells were progressing to a G2 block (Fig. 4A). The 3T3 cells transformed by polyoma, another DNA virus, and DNA distribution in treated MSV-NRK cells was the same as cells transformed by both SV40 and polyoma (Fig. 5B) showed that in the control (Fig. 4C). This suggests that MSV-NRK cells peaks at both G1 and G2 but with a significant content of S phase were not blocked at any specific point in the cycle.