Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
Nucleotide excision repair phenotype of human acute myeloid leukemia cell lines at various stages of differentiation
Introduction
Terminally differentiated cells do not replicate their genome, and might thus dispense with the energy expense required to repair it, provided that the small subset of genes that are active can be proficiently repaired. This is indeed what we and others have observed in several types of differentiated cells (reviewed in [1]), such as neurons. Among the many repair pathways, nucleotide excision repair (NER) appears to be especially modulated by differentiation: terminally differentiated neurons displayed a strong impairment in global genome repair (GGR), while still proficiently repairing their active genes [2]. The repair of the transcribed strand (TS) can be explained by the fact that NER is coupled to transcription, whereas to explain the proficient repair of the non-transcribed strand we postulated the existence of another sub-pathway of NER, which we called differentiation-associated repair (DAR) [2].
The present study was undertaken to answer the following two questions: (1) Does the NER impairment only happen in terminally differentiated cells, or can we observe a gradual decrease in GGR capability as differentiation progresses? (2) Can we observe DAR in cell types other than neurons, in particular in cells that have not yet reached the endpoint of their differentiation pathway?
To address these questions, we made use of a series of human acute myeloid leukemia (AML) cell lines, taken as representative of various steps of differentiation on the myelo-monocytic pathway. Human acute myeloid leukemia (AML), the most common leukemia type in adults, arises from neoplastic transformation in bone marrow cells. Based on the stage of differentiation that pluripotent stem cells have reached at the time of diagnosis, the traditional French–American–British classification (FAB) further categorized the heterogeneous AML into subtypes; e.g., along the monocyte-macrophage lineage, myeloblastic (M1, immature; M2, mature), promyelocytic (M3), myelomonocytic (M4) and monocytic (M5) leukemia subtypes were proposed based on morphological and cytochemical criteria [3]. AML cell lines corresponding to these subtypes have been established since late 1970s. Following upon discoveries of an array of inducers that trigger terminal differentiation in many of these cells [4], laboratories worldwide have used them as a model system for studying cellular maturation.
We selected four cell lines representative of various stages of differentiation: KG-1a, KG-1, HL-60 and THP-1 (Fig. 1). The KG-1 line is blocked in cell maturation at the myeloblast stage (M2) [5], but can be induced to differentiate into macrophage-like cells by treatment with phorbol esters (TPA). Because macrophage-like cells are adherent, it is very easy to obtain 100% pure cultures of differentiated cells, by just rinsing off the floating cells. A morphologically, cytochemically and immunologically less mature KG-1 variant cell line, designated KG-1a, was established in 1980 [6]. Contrarily to its parent cell line, KG-1a cannot be differentiated by TPA.
HL-60 represents a more differentiated promyelocyte stage (M3), and can also be differentiated into macrophage-like cells by treatment with TPA [7], as well as into granulocyte-like cells by treatment with either retinoic acid or dimethyl-sulfoxide (DMSO) [8]. For the purpose of this work, we only considered macrophage-like cells. As a note of caution it should be emphasized that, due to their extreme genomic instability, there exist many sub-lines of “HL-60 cells”, with widely different phenotypes [9]. In addition, phenotypes may vary with time if cells are kept in culture long enough. For instance, we had several occurrences of high passage number-HL-60 cells losing all NER capability. Western blot analysis revealed that they had lost one the NER genes, such as XPC, hHR23B, or XPG. The HL-60 sub-line used for the present study was obtained from ATCC (catalog number CCL-240), and we were careful to only use low (i.e. 10–20) passage numbers.
Finally, THP-1 represents the monocyte stage M5 and can also be differentiated into macrophage-like cells with TPA.
NER is a highly versatile repair system, capable of repairing a great variety of lesions, from UV-induced pyrimidine dimers, to bulky chemical adducts, to intra-strand crosslinks. This amazing feat is accomplished by a relatively small number of enzymes: the presence of a DNA lesion is detected by the recognition enzymes XPC, XPA, and for some lesions DDB. A denaturation bubble is then opened around the lesion with the help of the general transcription factor TFIIH, which contains two helicases of opposite polarities, XPB and XPD. The damaged strand is then incised on either side of the lesion, by XPG in 3′ and by the heterodimer ERCC1/XPF in 5′. The short oligonucleotide spanning the lesion is then displaced, and the resulting gap filled in by DNA polymerases delta or epsilon, and sealed by ligase I.
Two major sub-pathways of NER, transcription-coupled repair (TCR) and global genomic repair (GGR), have been identified and reviewed in [10]. Briefly, the operational definition of TCR is that certain lesions, such as CPDs, are removed faster from transcribed strands than from non-transcribed strands in active genes; GGR refers to the repair of the entire genome, including non-transcribed strands of active genes. As mentioned above, we have recently proposed the existence of yet another sub-pathway of NER, DAR, following upon our studies of CPD repair during terminal differentiation of the human neuroteratoma cell line NT2 and of primary neurons [1], [2]. DAR refers to the phenomenon that in certain cell types, when terminal differentiation is complete, there is a more rapid removal of certain lesions from non-transcribed strands of active genes in comparison with background GGR levels.
Section snippets
Cell culture and differentiation conditions
All cell lines were obtained from ATCC. KG-1a and KG-1 cells were grown in Iscove's modified Dulbecco's medium (IMDM, Gibco) supplemented with 20% fetal bovine serum and 0.15% NaHCO3; HL-60 and THP-1 cells were cultured in RPMI medium 1640 (Gibco) with 10% fetal bovine serum and 1 mM sodium pyruvate. Exponentially growing cells were uniformly pre-labeled for 10 days with [3H]-thymidine at a final concentration of 0.5 μCi/ml. For experiments involving terminally differentiated cells along the
Repair of UV-induced lesions in vivo
To appraise the NER capability of AML cell lines, we challenged them with 254 nm UV light, which allowed us to precisely control the amount of damage to their DNA, as well as the exact time of damage. At the wavelength used, UV light induces primarily two kinds of lesions in the DNA: a majority of CPDs and a lesser amount (generally 1/3 to 1/4) of (6-4)PPs [17]. Both lesions are characterized by the formation of covalent bonds between adjacent pyrimidines on the same DNA strand, and differ only
Discussion
The present study was undertaken mainly to answer two questions. The first one was whether there is a gradual drop in the efficiency of NER as differentiation progresses. This does not appear to be the case: CPDs were poorly repaired in all four naïve cell lines as well as in differentiated macrophage-like cells. The repair of (6-4)PPs was very efficient in all cell types, and was not significantly affected by differentiation. Only cisplatin intrastrand crosslinks were repaired less efficiently
Acknowledgments
We are indebted to Toshio Mori for providing the antibodies against CPD and (6-4)PPs, and to Stephen Lloyd for a generous supply of T4 endonuclease V. We thank Rick Wood, Steve Patrick and Silvia Tornaletti for advice on the cisplatin excision assay. This work was supported by an award from the Ellison Medical Foundation and a grant (CA77712) from the National Cancer Institute, NIH.
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