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Stochastic acquisition of a stem cell-like state and drug tolerance in leukemia cells stressed by radiation

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Abstract

A rare population of leukemia cells have the properties of leukemia stem cells (LSCs) and cause resistance to therapy, but their development is not clearly understood. In the current study, we show that a higher resistance to cytotoxic drug (Ara-C) can be developed in the subpopulation of promyelocytic leukemia cells that survived radiation treatment. These drug-tolerant leukemia cells (DTLs) are not observed immediately after radiation despite extensive genetic instability in the cells, but appear in 3 weeks of recovery culture. Moreover, when the single cell-derived clones were examined by clonal trafficking, no correlation between radio-resistant and chemo-resistant leukemic clones was detected, indicating that the resistance is developed by active acquisition of the resistance without clonal predisposition. Interestingly, the DTLs mimicked the characteristics of LSCs exhibiting leukemia-initiating activities and lower levels of reactive oxygen species or a higher level expression of bmi-1 as well as higher resistance to retinoic acid-induced differentiation compared to parental leukemic cells. These studies show that an active reacquisition of stem cell-like properties can occur in the leukemia cells to develop resistance to treatments and that such reacquisition process of leukemic cells occurs in a stochastic manner triggered by radiation stress on leukemic cells.

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Acknowledgments

This research was supported by Korea Science and Engineering Foundation (KOSEF) (Grant Code: 2008-05981) funded by the Korean Government (MEST) and National Agenda Project (NAP, 2009).

Conflict of interest

The authors have no conflict of interest to disclose.

Author information

Authors and Affiliations

  1. Catholic High-Performance Cell Therapy Center, College of Medicine, The Catholic University of Korea, 505, Banpo-Dong, Seocho-Ku, Seoul, 137-701, Republic of Korea

    Ga-Young Lee, Jae-Seung Shim, Joo-Young Jung & Il-Hoan Oh

  2. Catholic Stem Cell Transplantation Center, Seoul, Republic of Korea

    Bin Cho

  3. Department of Laboratory Medicine, Seoul National University, Seoul, Republic of Korea

    Dong-Soon Lee

Authors
  1. Ga-Young Lee
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  2. Jae-Seung Shim
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  3. Bin Cho
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Corresponding author

Correspondence to Il-Hoan Oh.

Electronic supplementary material

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Supplementary Figure 1. Kinetic changes in viability of HL-60 cells after irradiation or Ara-C treatment

(A): HL-60 cells (4 x 105 /ml) were 137Cs γ-ray irradiated at indicated dose of radiation (500 or 700cGy) (A), or treated with indicated concentrations (Left: 0, 20, 60, 80 µM, Right: 40 µM) of cytosine arabinoside (Ara-C) for 6 hrs (B), then viability was measured by trypan blue exclusion at each indicated time points. Results are expressed as the percentage of viability relative to the viability at the time of treatment (0 hrs). Shown are the mean ± SEM of viability (n = 3 for each). (TIFF 94 kb)

Supplementary Fig. 2 Genetic instability of leukemia cells exposed to radiation assessed by alkaline comet assay.

Un-irradiated or irradiated HL-60 cells were harvested at each indicated time points after irradiation (1hour, 1week or 3weeks after irradiation). Shown are the mean of the length of tail normalized to the control group (n=2 experiments). (TIFF 61 kb)

Supplementary Fig. 3 Leukemic colony forming activity of DTL and control leukemic cells.

For each assay, 1 x 102 cells were cultured in methocult H4230TM supplemented with cytokines. Secondary CFC assays were performed by replating aliquots of cells obtained by harvesting complete 10-day-old primary CFC cultures. Shown are the mean numbers of colonies ± SEM (n = 3 for each group). (TIFF 57 kb)

Supplementary Fig. 4 Expression level of bmi-1 and ROS levels in early point recovery culture. (A) Control leukemic cells and the cells at earlier (1week) point of recoveries were compared

for expression levels of bmi-1 by RT-PCR. Shown are representative plots (left) and relative expression levels in early recovery leukemic cells normalized to the levels in the control parental cells (right) (n= 2 for each group). (B) ROS levels were compared by staining each leukemic cells with DCF-DA (1 µM). Shown are the representative plots in flowcytometry (left) and mean (n = 2 for each group) of mean fluorescent intensity (MFI). (TIFF 99 kb)

Supplementary Fig. 5 Comparisons for expression level of bcl-2/bax in control and DTLs. (A):

DTLs and control leukemic cells were analyzed for expression of bcl-2 and bax by RT-PCR. The ratio of expression level (Bcl-2/Bax) relative to the control leukemic cells are shown with mean ± SEM (n=6) (P>0.05). (TIFF 55 kb)

Supplementary Fig. 6 Verification of Ara-C resistance in the leukemic cells survived from Ara-C.

HL-60 cells (5 x 105cells/ml) were plated in 24-well plate and Ara-C (or PBS for negative control) was added (final concentration; 40µM). 6 hrs after treatment, the cells were washed with PBS and re-plated in 24-well plate. 7 days later, the cells were treated with Ara-C (or PBS). The viabilities were then measured by trypan blue exclusion assay. Shown are the mean ± SEM (n = 3 for each group). (TIFF 63 kb)

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Lee, GY., Shim, JS., Cho, B. et al. Stochastic acquisition of a stem cell-like state and drug tolerance in leukemia cells stressed by radiation. Int J Hematol 93, 27–35 (2011). https://doi.org/10.1007/s12185-010-0734-2

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  • DOI: https://doi.org/10.1007/s12185-010-0734-2

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