Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 12 (9), e0185010
eCollection

A Novel Class of Chemicals That React With Abasic Sites in DNA and Specifically Kill B Cell Cancers

Affiliations

A Novel Class of Chemicals That React With Abasic Sites in DNA and Specifically Kill B Cell Cancers

Shanqiao Wei et al. PLoS One.

Abstract

Most B cell cancers overexpress the enzyme activation-induced deaminase at high levels and this enzyme converts cytosines in DNA to uracil. The constitutive expression of this enzyme in these cells greatly increases the uracil content of their genomes. We show here that these genomes also contain high levels of abasic sites presumably created during the repair of uracils through base-excision repair. We further show that three alkoxyamines with an alkyne functional group covalently link to abasic sites in DNA and kill immortalized cell lines created from B cell lymphomas, but not other cancers. They also do not kill normal B cells. Treatment of cancer cells with one of these chemicals causes strand breaks, and the sensitivity of the cells to this chemical depends on the ability of the cells to go through the S phase. However, other alkoxyamines that also link to abasic sites- but lack the alkyne functionality- do not kill cells from B cell lymphomas. This shows that the ability of alkoxyamines to covalently link to abasic sites is insufficient for their cytotoxicity and that the alkyne functionality may play a role in it. These chemicals violate the commonly accepted bioorthogonality of alkynes and are attractive prototypes for anti-B cell cancer agents.

Conflict of interest statement

Competing Interests: ASB is a member of AAAS and ASM. All authors are affiliated with Wayne State University, which could eventually benefit if a patent is granted on AA3 and related compounds. A patent regarding AA3 is pending (PCT/US2017/012279). This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Reaction of alkoxyamines with AP sites.
(A) The open-chain aldehyde form of an AP site in DNA reacts with an alkoxyamine (H2N-O-R). (B) Structures of different alkoxyamines used in this study.
Fig 2
Fig 2. Levels of genomic AP sites and expression genes responsible for the creation and repair of AP sites.
(A) Relative levels of AP sites in the DNA of B-NHL and non-B-NHL cells. The level of AP sites of each cell line is shown relative to the level in HeLa cells (set to 1). **** is P-value <0.0001, and *** is P-value <0.0005. In all cases, the mean and standard deviations are shown. (B) The expression of different genes in Daudi, Raji and HeLa cells relative to the β actin gene set at 100. “*” denotes undetectable expression.
Fig 3
Fig 3. Sensitivities of different cells to AA3.
(A) Killing of B-NHL cells by micromolar concentrations of AA3. (B) Killing of B-NHL cells by millimolar concentrations of AA3. (C) Comparison of AA3 sensitivities of Daudi cells, normal human B cells and primary human keratinocytes (HEKn). (D) Comparison of AA3 sensitivity of Daudi cells with non-B cell lines HeLa, MCF-7, MDA-MB 453, A549 and HEK293T. The data in panel A is from triplicates, while the data in the remaining panels is from six replicates. (E) Correlation between AP sites in B-NHL and other cell lines and killing by treatment with 5 mM AA3. These data are from Fig 2, and parts B and D of this figure. In all cases, the mean and standard deviations are shown.
Fig 4
Fig 4. Covalent binding of AA3 to genomic DNA in vivo.
Fluorescence intensity due to Cy5 bound to genomic DNA is shown. (A) Comparison of labeling of HeLa and Daudi cell DNA with AA3. The cells were treated with AA3 for indicated lengths of time and this was followed by DNA extraction and reaction with Cy5 azide. (B) Comparison of labeling of DNA from untreated cells with cells pretreated with AA3. Genomic DNA was isolated, labeled with ARP and bound to Cy5-Streptavidin. (C) Comparison of binding of AA3 to genomic DNA in dead or dying cells with live cells. Cells were treated with AA3, dead cells were separated from live cells, the genomic DNA was extracted and reacted with Cy5 azide. The data are from six replicates, and the mean and standard deviation are shown. ** represents P-value <0.005, n.s. is not significant.
Fig 5
Fig 5. Detection of γ-H2AX in Daudi cells following AA3 treatment.
(A) Daudi cells were stained with anti-γ-H2AX antibodies and visualized using immunofluorescence. Untreated cells are compared with AA3- or AA6-treated, or Phleomycin-treated cells. Representative images of cells stained with DAPI (blue), anti-γ-H2AX antibodies labeled with Cy3 (red) and the overlay of the two images are shown. (B) Quantification of γ-H2AX nuclear fluorescence intensity. The total number of cells used for quantification- Untreated, 74; AA6-treated, 70; AA3-treated, 184 and Phleomycin-treated, 122. Mean intensity and standard deviation for each cell type is shown.
Fig 6
Fig 6. Insensitivity of G1-arrested Daudi cells to AA3.
Daudi cells grown in normal growth media (-Mimosine), cells blocked in G1 using Mimosine treatment (+Mimosine) and cells released from Mimosine block (Mimosine release) were incubated with AA3 and the cell viability was determined. The numbers were normalized to the viability of cells without AA3 treatment (set to 100). The data are shown as the mean and standard deviation from six replicates (** represents P-value <0.005, n.s. is not significant).
Fig 7
Fig 7. Comparison of the sensitivity of a B-NHL cell line to different alkoxyamines.
(A) Comparison of sensitivity of Daudi cells to MX, ARP or AA3. (B) Reduction in cell growth following treatment of Daudi cells with MX, ARP and AA3. Cells were treated for 24 hr at indicated concentrations of chemicals. Horizontal dotted line represents the starting density of cells. “*” is P-value of <0.05 and “**” is P-value of <0.01. (C) Suppression of AA3 cytotoxicity for Daudi cells by pre-treatment of the cells with MX. (D) Comparison of cytotoxicities of different analogs of AA3 for Daudi cells. In all cases, the viability of untreated cells (NT) is set at 100% and the mean and standard deviation of six replicates are shown (** represents P-value <0.005, n.s. is not significant).
Fig 8
Fig 8. Effects of CRT0044876 on APE-1 and Raji cells.
(A) A synthetic oligomer containing a uracil was treated with Ung to create an AP site and the AP site cleaved by APE-1 in the presence or absence of the inhibitor CRT0044876 (100 μM). (B) Raji cells were treated with different concentrations of CRT0044876 dissolved in DMSO and the viability of the cells after 24 hr treatment is presented. “DMSO” represents viability of cells following treatment with 1% DMSO alone.

Similar articles

See all similar articles

Cited by 3 PubMed Central articles

References

    1. Bransteitter R, Pham P, Scharff MD, Goodman MF. Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase. Proc Natl Acad Sci U S A. 2003;100(7):4102–7. Epub 2003/03/26. doi: 10.1073/pnas.0730835100 - DOI - PMC - PubMed
    1. Chaudhuri J, Tian M, Khuong C, Chua K, Pinaud E, Alt FW. Transcription-targeted DNA deamination by the AID antibody diversification enzyme. Nature. 2003;422(6933):726–30. Epub 2003/04/15. doi: 10.1038/nature01574 - DOI - PubMed
    1. Dickerson SK, Market E, Besmer E, Papavasiliou FN. AID mediates hypermutation by deaminating single stranded DNA. The Journal of experimental medicine. 2003;197(10):1291–6. doi: 10.1084/jem.20030481 - DOI - PMC - PubMed
    1. Muramatsu M, Sankaranand VS, Anant S, Sugai M, Kinoshita K, Davidson NO, et al. Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells. The Journal of biological chemistry. 1999;274(26):18470–6. - PubMed
    1. Sohail A, Klapacz J, Samaranayake M, Ullah A, Bhagwat AS. Human activation-induced cytidine deaminase causes transcription-dependent, strand-biased C to U deaminations. Nucleic Acids Res. 2003;31(12):2990–4. Epub 2003/06/12. - PMC - PubMed

MeSH terms

Feedback