Print version ISSN 1516-8484
Rev. Bras. Hematol. Hemoter. vol.33 no.5 São Paulo Oct. 2011
Jessica Romy TsudaI,II; Rosimeire SegatoI; Waldênia BarbosaIII; Marília de Arruda Cardoso SmithII; Spencer Luiz Marques PayãoI,IV
IGenetics Laboratory, Hemocentro, Faculdade de Medicina de Marília - FAMEMA, Marília, SP, Brazil
IIDepartment de Morphology, Universidade Federal de São Paulo - UNIFESP, São Paulo, SP, Brazil
IIIHematology Department, Faculdade de Medicina de Marília - FAMEMA, Marília, SP, Brazil
IVUniversidade do Sagrado Coração - USC, Bauru, SP, Brazil
BACKGROUND: Recently, the importance of cytogenetics has grown in the diagnosis, prognosis and treatment of leukemias and myelodysplastic syndromes. 5-azacytidine is a drug that has well-known cytogenetical effects and is approved in the treatment of myelodysplastic syndromes. To date, no studies have been performed to evaluate the impact of 5-azacytidine on the chromosomes of patients with hematological neoplasias. This study aimed to investigate the effects of 5-azacytidine on chromosomes of patients with different hematological malignancies using G-band analyses to identify possible cytogenetical alterations.
METHODS: The peripheral blood of 18 patients with hematological malignancies and 18 controls was collected in heparinized tubes. 5-azacytidine was added, at a final concentration of 10-5M, to cultures 7 hours prior to harvest.
RESULTS: Uncoiled centromeric/pericentromeric heterochromatin of chromosomes-1, 9 and 16 occurred more frequently in the patients than in controls. This higher frequency of uncoiled heterochromatin was statistically significant (p-value = 0.004) for chromosome-9. Conversely, we observed that the fragile site at 19q13 was more frequent in controls (p-value = 0.0468).
CONCLUSIONS: The results of this study suggest that satellite sequences, located in the heterochromatin of chromosome-9, are hypomethylated in hematological malignancies. This hypomethylation may contribute to the disease, activating transposable elements and/or promoting genomic instability, enabling the loss of heterozygosity of important tumor suppressor genes. An investigation of the 19q13 region may help to understand whether or not the predominant occurrence of the fragile site at 19q13 in controls is due to hypermethylation of this region.
Keywords: Cytogenetics; Azacitidine; Heterochromatin
Leukemias and myelodysplastic syndromes (MDS) are both clonal hematopoietic stem cell disorders, but while MDS is characterized by ineffective hematopoiesis, peripheral cytopenias and cytological atypia in one or more myeloid cell lineages, leukemias involve overproliferation of hematopoietic stem cells which when added to their failure to differentiate leads to an accumulation of non-functional cells, known as blasts, in the bone marrow and the peripheral blood.(1-4) About 30% of MDS cases evolve to acute myeloid leukemia (AML), thus MDS is considered a pre-leukemia disease.(5) Aberrant karyotypes are found in 30-50% of MDS, 40% of AML, 64-85% of adult acute lymphoid leukemia (ALL), 60-69% of pediatric ALL and in 95% of chronic myeloid leukemia (CML).(2-5)
Pretreatment cytogenetic findings have repeatedly proved to be among the most important independent prognostic factors for AML and ALL.(4) Chromosomal analyses are important prognostic markers; this is one of the parameters evaluated by the International Prognostic Scoring System (IPSS), the gold standard for risk assessment of patients with de novo MDS.(1)
DNA methylation is an epigenetic process, meaning it has heritable information other than the DNA sequence involved in gene expression.(6,7) This process is essential for embryonic development, tissue differentiation, X chromosome inactivation in females and imprinting.(7,8) Epigenetic alterations have gained increasing recognition as important participants in tumor development and progression.(9)Abnormal DNA methylation patterns have been recognized in cancer cells for over two decades.(10) It is well established that although the genome as a whole is hypomethylated in cancer, CpG islands located in promoter regions of certain genes are found hypermethylated.(9,11-16) In experimental settings, demethylating agents are capable of reactivating tumor suppressor genes silenced by promoter hypermethylation.(14) 5-azacytidine (5-AZC) is a cytidine analog, in which a nitrogen atom replaces the carbon in the 5 position of the pyrimidine ring. As a result, when 5-AZC is incorporated into DNA, it cannot be methylated.(17) Low doses of 5-AZC induce DNA demethylation in malignant myeloid cells. The use of 5-AZC in clinical trials yielded positive results and so this was the first drug to be approved by the US Food and Drug Administration (FDA) for the treatment of MDS.(18)
When added into human lymphocyte cultures, 5-AZC is known to cause uncoiling of the centromeric/ pericentromeric heterochromatic regions (Figure 1A) of chromosomes 1, 9, 15, 16 and Y, allowing associations between these under-condensed regions (Figure 1B), which occurs during the interphase.(19,20) Cytogenetically, this demethylating agent also induces the formation of the fragile sites at 1q42 and 19q13 (Figure 1C) and, after 2 cycles of replication, it provokes differential under-condensation of sister-chromatids. (21,22)
In spite of the central role played by cytogenetics in the diagnosis, prognosis and treatment choice of leukemias and MDS, and of 5-AZC being a drug of well reported cytogenetical effects used to treat these diseases, no studies have been performed to evaluate the impact of 5-AZC on chromosomes of patients diagnosed with hematological neoplasias.
This study aimed to investigate the effects of the demethylating agent, 5-AZC, on chromosomes of patients with different hematological malignancies. In addition, Gband analyses were performed to detect possible chromosomal rearrangements in the peripheral lymphocytes of patients.
Heparinized peripheral blood samples from 13 female and 5 male patients with hematological malignancies were obtained at diagnosis ofmalignancies in the Hematology Department of FAMEMA (Faculdade de Medicina de Marília, SP, Brazil). The peripheral blood of 18 individuals, without history of neoplasias, matched for age (± 5 years) and gender to the patients was also collected and used as a control group. All subjects gave their informed consent to participate and this study which was approved by the local Ethics Committee.
Blood culture and 5-azacytidine treatment
Blood cells were seeded in RPMI 1640 medium supplemented with 10% fetal calf serum and incubated for 72 hours at 37ºC in a 5% CO2 atmosphere. 5-AZC was added to the culture 7 hours prior to harvest at a final concentration of 10-5 M. Harvesting was performed as described by the AGT Cytogenetics Laboratory Manual.(23)
Slide preparation and staining techniques
Slides were prepared following conventional techniques. Slides of 5-AZC-treated cultures were stained with 5% Giemsa for 8 minutes. Non-treated cultures of the patients were stained according to the G-band technique as described by the AGT Cytogenetics Laboratory Manual.(23)
One hundred Giemsa-stained 5-AZC-treated metaphases were analyzed for each subject. The parameters evaluated were: 1) the number of under-condensations of the heterochromatin of chromosomes 1, 9 and 16, 2) the occurrence of the fragile sites at 1q42 and 19q13 or other chromosomal lesions and 3) the presence of associations between uncoiled regions. Analyses were performed in a blind manner. Ten G-band metaphases of each patient were analyzed in order to investigate possible chromosomal alterations in these individuals.
The Mann-Whitney test was used to analyze the number of uncoiled heterochromatic regions of the human chromosomes 1, 9 and 16 and the number of associations between uncoiled regions and the Poisson test was used to analyze the fragile sites at 1q42 and 19q13. A level of significance was adopted for a p-value = 0.05 (Bioestat 5 software).
Through G-band analyses, the only consistent chromosomal abnormality found in patients was the Philadelphia chromosome in all 3 patients diagnosed with CML.
Patients 4 and 9 had Down's syndrome and therefore an extra chromosome 21 was observed in all metaphases of these individuals.
Our results showed that the centromeric/ pericentromeric heterochromatin of chromosomes 1, 9 and 16 were more frequently uncoiled in the group of patients compared to the control group and this higher frequency of uncoiled heterochromatin was statistically significant (p-value = 0.004) for chromosome 9 (Table 1).
The cytogenetical effects of 5-AZC were first described in 1976(19-22) and its use in the treatment of MDS was approved by the US FDA in 2004.(24,25) Nevertheless, to date, studies evaluating the effects of 5-AZC on chromosomes of patients with hematological malignancies are unavailable in the literature.
Recently, cytogenetic analyses have gained importance in the diagnosis, prognosis and treatment choice for hematological malignancies. In the present study, the effects of exposure to 5-AZC were observed on the chromosomes of patients newly diagnosed with hematological malignancies.
The low incidence of cytogenetical abnormalities in the patients could be due to the fact that the samples were collected from peripheral blood instead of bone marrow. This finding suggests that the heterochromatic region of chromosome 9 is hypomethylated in patients, because it is more sensitive to the drug. The results are in accordance to what is described in the literature regarding neoplasias and hypomethylation.
Heterochromatin located in the centromeric/ pericentromeric region of chromosomes 1, 9 and 16 is mainly constituted by repetitive sequences known as satellites. The centromeric regions are rich in satellite a (Sat a), whereas the pericentromeric regions of chromosomes 1 and 16 are mainly constituted by satellite 2 (Sat 2) and of chromosome 9 by satellite 3 (Sat 3). In normal tissue, those regions are found heavily methylated.(26,27) It is possible that the higher frequency of uncoiled centromeric/pericentromeric heterochromatin of chromosome 9, observed in the patients, reflects alterations in the patterns of Sat 3 methylation in these individuals. On studying urothelial carcinoma, Nakagawa et al.(28) observed loss of heterozygosity (LOH) of chromosome 9 in more than half of the cases (52%) and, in the cases where LOH of chromosome 9 was detected, rather large regions of 9p and/or 9q were lost. DNA hypomethylation of Sat 2 and Sat 3 was significantly correlated with LOH of chromosome 9 in this neoplasia. Thus, the study concluded that DNA hypomethylation of pericentromeric satellite regions may participate in the development and progression of urothelial carcinomas by inducing loss of heterozygosity of chromosome 9. It is possible that hypomethylation of satellite sequences can contribute to the occurrence and progression of hematological malignancies as well. Fragile sites at 1q42 and 19q13 were more common in the control group than in patients, but the difference was only statistically significant (p-value = 0.0468) for 19q13 (Table 2).
One possible explanation for this finding is that the 19q13 region is hypermethylated in patients and, therefore, is less sensitive to the action of 5-AZC. We can speculate that, in the 19q13 region, there is a tumor suppressor gene whose silencing through methylation favors the development of the neoplasia. The LOH of the 19q13 region is commonly found in neuroblastomas and gliomas, suggesting the existence of an important tumor suppressor gene in this region involved in the development of these cancers. One candidate gene is the epithelial membrane protein 3 (EMP3).(29)
Nevertheless, studies associating hypermethylation of either the EMP3 gene or the 19q13 band and hematological malignancies were not found in the literature. The induction of hypomethylation using 5-AZC provokes translocations between satellite regions in the second cell generation, probably resulting from the somatic associations observed between regions in the first cell generation.(30) Associations involving the uncoiled regions of chromosomes 1, 9 and 16 did not statistically differ between patients and the control group. However, patients 3, 5, 7 and 17 presented respectively 16, 9, 14 and 10 associations whereas the highest number of associations found in the control group was 6 (Table 3). The high frequency of associations exhibited by these 4 patients could be indicative that the satellite regions of these individuals are more unstable.
As previously mentioned, in cancer, the genome as a whole is hypomethylated and the loss of methyl groups occurs mainly in the body of genes and in the repetitive sequences, such as the sequences present in the heterochromatic region of chromosomes 1, 9 and 16. Overall hypomethylation has the potential to contribute to a malignant phenotype through chromosomal instability, reactivation of transposable elements, and loss of normal gene imprinting patterns.(14,26) The results of this study showed that the centromeric/pericentromeric heterochromatin of chromosomes 1, 9 and 16 appeared more frequently uncoiled in the group of patients compared to the control group and this higher frequency of uncoiled heterochromatin was statistically significant (p-value = 0.004) for chromosome 9 (Table 1). This finding suggests that the heterochromatic region of chromosome 9 is hypomethylated in patients because it is more sensitive to the drug. Results suggest that the satellite DNA of chromosome 9 is hypomethylated in patients with different hematological malignancies. It is possible that this hypomethylation contributes to the pathogenesis of these neoplasias by activating parasite sequences and/or promoting chromosomal instability. In addition, the higher frequency of the fragile site at 19q13 in the control group may indicate that that region is hypermethylated in patients; nonetheless molecular studies searching for candidate tumor suppressor genes involved in the development and/or progression of hematological malignancies located in the 19q13 band are necessary in order to prove this finding. The increased occurrence of associations between the uncoiled regions of chromosomes 1, 9 and 16 observed in 4 patients could be a sign that the satellite DNA in these individuals is unstable.
The authors would like to thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). This research was supported by Faculdade de Medicina de Marília, FAMEMA.
1. Valent P, Horny HP, Bennett JM, Fonatsch C, Germing U, Greenberg P, et al. Definitions and standards in the diagnosis and treatment of the myelodysplastic syndromes: Consensus statements and report from a working conference. Leuk Res. 2007;31(6):727-36. [ Links ]
2. Pui CH, Jeha S. New therapeutic strategies for the treatment of acute lymphoblastic leukaemia. Nat Rev Drug Discov. 2007;6(2):149-65. [ Links ]
3. Melo JV, Barnes DJ. Chronic myeloid leukaemia as a model of disease evolution in human cancer. Nat Rev Cancer. 2007;7(6):441-53. [ Links ]
4. Mrózek K, Heerema NA, Bloomfield CD. Cytogenetics in acute leukemia. Blood Rev. 2004;18(2):115-36. [ Links ]
5. Galili N, Cerny J, Raza A. Current treatment options: impact of cytogenetics on the course of myelodysplasia. Curr Treat Options Oncol. 2007;8(2):117-28. [ Links ]
6. Fandy TE, Herman JG, Kerns P, Jiemjit A, Sugar EA, Choi S, et al. Early epigenetic changes and DNA damage do not predict clinical response in an overlapping schedule of 5-azacytidine and entinostat in patients with myeloid malignancies. Blood. 2009;114(13):2764-73. [ Links ] Comment in: Blood. 2009;114(13):2569-70. [ Links ]
7. Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet. 2003;33(Suppl):245-54. [ Links ]
8. Panning B, Jaenisch R. DNA hypomethylation can activate Xist expression and silence X-linked genes. Genes Dev. 1996;10(16): 1991-2002. [ Links ]
9. Rush LJ, Plass C. Alterations of DNA methylation in hematologic malignancies. Cancer Lett. 2002;185(1):1-12. [ Links ]
10. Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature. 1983;301(5895):89-92. [ Links ]
11. Esteller M. Profiling aberrant DNA methylation in hematologic neoplasms: a view from the tip of the iceberg. Clin Immunol. 2003;109(1):80-8. [ Links ]
12. Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128(4):683-92. [ Links ]
13. McCormack SE, Warlick ED. Epigenetic approaches in the treatment of myelodysplasic syndromes: clinical utility of azacitidine. Onco Targets Ther. 2010;3:157-65. [ Links ]
14. Galm O, Herman JG, Baylin SB. The fundamental role of epigenetics in hematopoietic malignancies. Blood Rev. 2006;20(1):1-13. [ Links ]
15. De Carvalho DD, You JS, Jones PA. DNA methylation and cellular reprogramming. Trends Cell Biol. 2010;20(10):609-17. [ Links ]
16. Miranda TB, Jones PA. DNA methylation: the nuts and bolts of repression. J Cell Physiol. 2007;213(2):384-90. [ Links ]
17. Haaf T. The effects of 5-azacytidine and 5-azadeoxycytidine on chromosome structure and function: implications for methylation-associated cellular processes. Pharmacol Ther. 1995;65(1):19-46. [ Links ]
18. Boultwood J, Wainscoat JS. Gene silencing by DNA methylation in haematological malignancies. Br J Haematol. 2007;138(1):3-11. [ Links ]
19. Schmid M, Haaf T, Grunert D. 5-Azacytidine-induced undercondensations in human chromosomes. Hum Genet. 1984;67(3):257-63. [ Links ]
20. Haaf T, Steinlein K, Schmid M. Preferential somatic pairing between homologous heterochromatic regions of human chromosomes. Am J Hum Genet. 1986;38(3):319-29. [ Links ]
21. Schmid M, Ott G, Haaf T, Scheres JM. Evolutionary conservation of fragile sites induced by 5-azacytidine and 5-azadeoxycytidine in man, gorilla, and chimpanzee. Hum Genet. 1985;71(4):342-50. [ Links ]
22. Haaf T, Ott G, Schmid M. Differential inhibition of sister chromatid condensation induced by 5-azadeoxycytidine in human chromosomes. Chromosoma. 1986;94(5):389-94. [ Links ]
23. Barch MJ, Knutsen T, Spurbeck, JL, editors. The AGT cytogenetics laboratory manual. 3rd ed. New York: Lippincott-Raven;1977. [ Links ]
24. Kaminskas E, Farrell AT, Wang YC, Sridhara R, Pazdur R. FDA drug approval summary: azacitidine (5-azacytidine, Vidaza) for injectable suspension. Oncologist. 2005;10(3):176-82. [ Links ]
25. Muller A, Florek M. 5-Azacytidine/Azacitidine. Recent Results Cancer Res. 2010;184:159-70. [ Links ]
26. Grønbaek K, Hother C, Jones PA. Epigenetic changes in cancer. APMIS. 2007;115(10):1039-59. [ Links ]
27. Ehrlich M. DNA methylation in cancer: too much, but also too little. Oncogene. 2002;21(35):5400-13. [ Links ]
28. Nakagawa T, Kanai Y, Ushijima S, Kitamura T, Kakizoe T, Hirohashi S. DNA hypomethylation on pericentromeric satellite regions significantly correlates with loss of heterozygosity on chromosome 9 in urothelial carcinomas. J Urol. 2005;173(1):243-6. [ Links ]
29. Alaminos M, Dávalos V, Ropero S, Setién F, Paz MF, Herranz M, et al. EMP3, a myelin-related gene located in the critical 19q13.3 region, is epigenetically silenced and exhibits features of a candidate tumor suppressor in glioma and neuroblastoma. Cancer Res. 2005;65(7):2565-71. [ Links ]
30. Kokalj-Vokac N, Almeida A, Viegas-Péquignot E, Jeanpierre M, Malfoy B, Dutrillaux B. Specific induction of uncoiling and recombination by azacytidine in classical satellite-containing constitutive heterochromatin. Cytogenet Cell Genet. 1993;63 (1):11-5. [ Links ]
Spencer Luiz Marques Payão
Laboratório de Genética, Hemocentro, FAMEMA
Rua Lourival Freire, 240, Bairro Fragata
17519-050 - Marília, SP, Brazil
Phone: 55 14 3402-1856
Conflict-of-interest disclosure: The authors declare no competing financial interest