Radiolabeling of DNA can induce its fragmentation in HL-60 human promyelocytic leukemic cells

doi:10.1016/0014-4827(92)90027-6

Experimental Cell Research

Volume 203, Issue 2, December 1992, Pages 495-498

https://doi.org/10.1016/0014-4827(92)90027-6 Get rights and content

Abstract

Incorporation of radiolabeled thymidine is commonly used to investigate DNA damage. Using a filter-binding assay, we observed that the addition of various doses of [methyl-³H]thymidine (0.2 and 2 μCi/ml) or [2-¹⁴C]thymidine (0.02 and 0.2 μCi/ml) in the culture medium for 2 days, a standard method for cell-labeling, induces DNA fragmentation in HL-60 human promyelocytic cells. This effect was dose- and time-dependent and the DNA fragments were not protein-linked since the levels of DNA fragmentation were identical in the presence and in the absence of proteinase K (0.5 mg/ml). Radiolabeled thymidine-induced DNA fragmentation was associated with an inhibition of cell growth, but cells remained able to exclude trypan blue, suggesting that plasma membrane integrity was conserved, except at very high doses of [methyl-³H]thymidine (2 μCi/ml). By agarose-gel electrophoresis, the DNA-fragmentation was demonstrated to be internucleosomal with a typical ladder pattern. Addition of unlabeled thymidine to the culture medium prevented DNA fragmentation in a dose-dependent manner, indicating that radiolabeled thymidine incorporation in DNA was directly responsible for DNA fragmentation. We conclude that radiolabeling of DNA using thymidine incorporation can induce DNA fragmentation in some cell lines such as HL-60. This observation must be taken into account in methods using radiolabeling to study DNA damage in these cells.

References (21)

M.J. Arends et al.
Int. Rev. Exp. Pathol
(1991)
M. Skalka et al.
FEBS Lett
(1976)
K.W. Kohn
Pharmacol. Ther
(1991)
M.C. Elia et al.
Pharmacol. Ther
(1991)
P. Pantazis et al.
Dev. Biol
(1981)
J.F.R. Kerr et al.
Br. J. Cancer
(1972)
M.J. Arends et al.
Am. J. Pathol
(1990)
S.V. Lennon et al.
Cell. Prolif
(1991)
R. Bertrand et al.
Cancer Res
(1991)
S.J. Martin et al.
Int. J. Radiat. Biol
(1991)

There are more references available in the full text version of this article.

Cited by (24)

Methods Utilized in the Study of Apoptosis
1997, Advances in Pharmacology
The growing awareness of the frequency and importance of physiological cell death (PCD) has resulted from two related developments, an increasing understanding of the biochemistry of PCD and a utilization of that understanding to develop sensitive methods for detecting PCD. The realization that this form of cell death is generally accompanied by internucleosomal DNA cleavage led to a variety of techniques for detecting this process, including DNA electrophoresis, flow cytometry, filtration assays, and in situ nick end labeling. Recent studies have demonstrated that PCD is accompanied by loss of phospholipid asymmetry across the plasma membrane as well as activation of various proteases, particularly members of the interleukin-1/3 converting enzyme (ICE) family. This chapter briefly describes the biochemical basis for each of these techniques and then attempts to summarize the strengths and weaknesses of each approach. In this regard Inhibitors of proteases (and other enzymes) are potentially useful tools when employed properly. Because the selectivity of these inhibitors is not fully known, however, experiments must be carefully designed, suitably controlled, and cautiously interpreted. Moreover, multiple alternative techniques (including antisense approaches and genetic knockouts) must be employed to confirm or refute conclusions suggested by inhibitor studies.
The effect of lovastatin on [<sup>3</sup>H]thymidine uptake in HTC-4 and LLC-L1 tumor cells
1996, Journal of Surgical Research
Lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, has been shown to inhibit thein vitroandin vivogrowth of a number of different cell lines. However, the mechanism by which lova- statin exerts its effect is not clear. In this experiment, we investigated the effect of lovastatin on the incorporation of [³H]thymidine by Hepatoma Tissue Culture-4 (HTC-4) and Lewis Lung Carcinoma L-1 (LLC-L1) tumor cells. Tumor cells were grown under standard conditions and treated with four different concentrations of lovastatin. Cell growth was evaluated by daily hemacytometer cell counts. On Day 4, the plates were pulsed with 10 μCi [³H]thymidine. After 24 hr, the plates were harvested and [³H]thymidine incorporation was measured by scintillation counting. Lova- statin inhibited both HTC-4 and LLC-L1 cell growth in a dose-dependent manner. At the highest lovastatin dose, both LLC-L1 and HTC-4 cell growth was slowed to less than 15% of control. Remarkably, however, both cell lines showed a paradoxical, dose related, increase in [³H]thymidine uptake. Cell cycle analysis using flow cytometry was performed on Day 5 in the LLC-L1 cell line. As the lovastatin concentration increased, a lower percentage of cells was found in the G₁phase of the cell cycle and a higher percentage of cells was found in the S and G₂phases. These findings suggest that these tumor cells are undergoing brisk DNA repair or that lovastatin is more effectively blocking cell division than cellular DNA replication.
Elevated levels of wild-type p53 induced by radiolabeling of cells leads to apoptosis or sustained growth arrest
1995, Current Biology
Background: The tumor suppressor protein p53 regulates progression through the checkpoint between the G1 and S phases of the cell cycle in response to radiationor drug-induced DNA damage. We have examined potential p53-mediated effects of metabolically labeling cultured mammalian cells with [³⁵S]methionine and [³H]thymidine, methods that are commonly used to study the biochemical properties, synthesis, processing and degradation of proteins and the replication of DNA in proliferating cells.
Results Wild-type p53 protein concentrations rapidly increased to high levels following metabolic radiolabeling of cells, as determined by four distinct assays. The increased concentration of wild-type p53 resulted in apoptosis of normal human peripheral blood lymphocytes and of murine T-cell acute lymphoblastic leukemia cells. In leukemia cells containing no p53, or only mutant p53 alleles, p53 protein levels were not increased and the cells did not undergo apoptosis in response to radioactive labeling. Radiolabeling of human diploid fibroblasts resulted in a prolonged growth arrest that was maintained for nearly three weeks.
Conclusion The results of experiments employing radiolabeling techniques to characterize various aspects of cellular physiology may be seriously influenced by the induction of aberrant cell-cycle arrest and/or apoptosis mediated by wild-type p53. Furthermore, our observations suggest that stabilization of wild-type p53 in response to irradiation may not act primarily to facilitate the repair of DNA damage by inducing a transient G1-phase arrest, but rather to ensure genetic stability through sustained cell-cycle arrest or apoptotic death of the damaged cells.
Phenomena of spontaneous proliferation, differentiation, and apoptosis in primary culture of leukemic lymphocytes
1994, Experimental Cell Research
The proliferative capacity of lymphocytes from peripheral blood of bovine with chronic lymphocytic leukemia (CLL) in vitro was investigated. We have shown earlier that CLL cells spontaneously proliferate in serum-free medium in the absence of added growth factors and mitogenic stimulation; autocrine growth factors provide the growth-initiating signal for CLL cells. The results of the present study showed that bovine serum albumin or fetal calf serum greatly enhanced the number of CLL cells incorporating [³H]thymidine. Although some CLL cells proceeded through more than one cell cycle, proliferation of CLL cells in culture was temporary. On the other hand, it was shown that CLL cells differentiated spontaneously in culture. This differentiation was characterized by the appearance of plasmacytoid cells possessing cytoplasmic immunoglobulins that coincided with the cessation of cell proliferation. Moreover, together with spontaneous proliferation and differentiation, the phenomenon of programmed cell death (apoptosis) was found, as was evidenced by the appearance of apoptotic bodies as well as DNA fragmentation. The findings indicate that the loss of proliferative potential of CLL cells in culture may be a consequence of their differentiation and/or apoptosis in vitro. CLL cells, with an autotrine growth mechanism, spontaneous differentiation, and apoptosis in vitro, provide a new model system for studies of the relationship between cellular proto-oncogene expression and inhibition of growth and/or induction of differentiation.
Visualization of dna replication in single chromosome by stable isotope labeling
2021, Cell Structure and Function
Pulse-chase analysis of procollagen biosynthesis by azidohomoalanine labeling
2014, Connective Tissue Research

View all citing articles on Scopus

²: Dr Eric Solary was supported by a grant from the Ligue Nationale Contre le Cancer (France) and from the North Atlantic Treaty Organization.

View full text

Short noteRadiolabeling of DNA can induce its fragmentation in HL-60 human promyelocytic leukemic cells

Abstract

Int. Rev. Exp. Pathol

FEBS Lett

Pharmacol. Ther

Pharmacol. Ther

Dev. Biol

Br. J. Cancer

Am. J. Pathol

Cell. Prolif

Cancer Res

Int. J. Radiat. Biol

Short note
Radiolabeling of DNA can induce its fragmentation in HL-60 human promyelocytic leukemic cells