Cancer Genetics and Cytogenetics 185 (2008) 114e116

Letter to the Editor

Mutational analysis of the CITED4 gene in glioblastomas

Allelic losses at 1p appear as a characteristic feature of glial tumors (glioblastoma, astrocytoma, oligodendroglioma, and mixed forms) and, together with 19q losses, represent a prognostic parameter predictive for chemosensitivity and survival primarily in anaplastic oligodendrogliomas [1e4]. Although several genes located at 1p ( p73, CAMTA1, p18ink4c, hRAD54, Patched2, Riz1, KIF1b) have been analyzed previously for inactivating mutations related to glioma development, no specic candidate genes have been identied [5e10]. CITED4 (CREB-binding protein/ p300-interacting transactivator with E/D-rich tail 4) is located at 1p34wp35 and encodes a 184eamino acid protein [11]. It is a member of the CITED family, which includes four identied genes, although only three of them are present in mammals (including humans): CITED1, 2, and 4 [12]. All family members share the presence of the CITED domain, which is able to interact with CBP (CREB-binding protein) and p300 [13]. These proteins are transcriptional co-activators that act in two ways to increase gene transcription: (1) by binding transcription factors with RNA polymerase II and (2) by acting like acetyltransferases [14]. Proteins CBP and p300 are able to act on the nucleosome [15]. CITED4 generally has a nuclear location, but cytoplasmatic translocation or loss of nuclear expression has been observed in breast cancer development, in which case it might represent a prognostic marker [13]. Therefore, CITED4 has been proposed as a candidate gene involved in neoplasms characterized by 1p loss. Tews and co-workers [16] recently reported on mutational (in 45 samples) and methylation (in 62 samples) studies of this gene. This glioma series primarily included tumors with major oligodendroglial components, and 15 different CITED4 polymorphisms, mostly single nucleotide exchanges, but no mutations were identied. In parallel, aberrant methylation of the CITED4-associated CpG island was primarily found in oligodendrogliomas with 1p/19q losses that generally showed at least 50% CITED4 reduced expression [16]. Although less frequent than in oligodendroglial tumors, allelic losses at 1p have also been described in astrocytomas (lowgrade and anaplastic) and glioblastomas [1,17], the most malignant form of glial neoplasms. Thus, we performed a mutational study of CITED4 in a series of 24 glial tumors (22 primary glioblastomas, 1 low-grade astrocytoma, and its recurrent secondary glioblastoma) using polymerase chain reaction/single-strand conformation polymorphism
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(PCR/SSCP) methods. To detect sequence changes, we used overlapping fragments of the entire coding region as described [16], and the primer sequences were as follows: 5-gggccaagacctagatgcag-3 and 5-aaaccaaacccgactg gtg-3 [229ebase pair (bp) amplicon]; 5-cgcaaggtgcgcagta gt-3 and 5-ctctgcaccaggcggtag-3 (229-bp amplicon); 5-c atggccgaccacctgat-3 and 5-gaaagggctggaaggaggac-3 (230-bp amplicon); 5-ctaccgcctggtgcagag-3 and 5-agtccg agaagcagtcgaac-3 (488-bp amplicon); and 5-catggacgccg aactcatc-3 and 5-agtcgggccctttctcctct-3 (212-bp amplicon). PCR/SSCP were performed in standard conditions, as we reported for mutational studies of other genes [5,6,9]. PCR products were bi-directionally sequenced using cycle sequencing (BigDye cycle sequencing kit; Applied Biosystems, Foster City, CA) and an ABI PRISM 377 semi-automated DNA sequencer (Applied Biosystems). Sequencing analysis of the PCR products allowed identication of six polymorphic sequence changes (Table 1), ve of them corresponding to those previously described by Tews and co-workers [16] and named CITED4 polymorphisms 1, 8, 9, 10, and 11 in their report. One additional change, not described previously, was also identied: NM_133467.2:c.197C / T(Ala66Ala). Table 2 shows the allelic frequencies detected for each polymorphic change in all 24 glioblastomas and the corresponding frequencies in the constitutional DNA in 12 cases for which peripheral blood lymphocyte DNA was available. Accordingly, in agreement with the previous data from oligodendrogliomas [16], the glioblastomas we studied did not show somatic CITED4 mutations potentially related to tumor development. According to Tews and co-workers [16], the signicant lower CITED4 mRNA expression in gliomas is linked to promoter hypermethylation so that complete CITED4 inactivation would be the result of the loss of one allele and hypermethylation of the other allele. This
Table 1 CITED4 polymorphisms identied in 24 glial (astrocytic) tumors Polymorphism no. according to Tews et al. [16] 1 8 9 10 11 Not reported previously Sequence change NC_000001:g.40997143 G / A NM_133467.2:c.72 G / T (no change) NM_133467.2:c.95 T / A (Leu32Gln) NM_133467.2:c.170 G / C (Arg57Pro) NM_133467.2:c.173A / G (Gln58Arg) NM_133467.2:c.197C / T (no change)

Letter to the editor / Cancer Genetics and Cytogenetics 185 (2008) 114e116 Table 2 CITED4 allele frequencies in tumor and constitutional DNA of 6 polymorphic changes identied in 24 glial (astrocytic) tumors Polymorphism Nucleotide Nucleotide Tumoral no. according position DNA frequency to Tews et al. [16] n Z 24 1 261 GG AA GA GG TT GT TT AA TA CC GG CG AA GG AG CC TT CT 11/24 1/24 12/24 19/24 -/24 3/24 23/24 -/24 1/24 -/24 23/24 1/24 23/24 -/24 1/24 22/24 -/24 2/24 Constitutional DNA frequency n Z 12 5/12 e/12 7/12 11/12 e/12 1/12 12/12 e/12 e/12 e/12 12/12 e/12 12/12 e/12 e/12 12/12 e/12 e/12

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Alberto Isla Cacilda Casartelli Juan A. Rey* Unidad de Investigacion Hospital Universitario La Paz Paseo Castellana 261 28046 Madrid, Spain *Corresponding author E-mail address: jarey.hulp@salud.madrid.org (J. A. Rey)

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References
[1] Bello MJ, Vaquero J, de Campos JM, Kusak ME, Sarasa JL, Saez J, Pestana A, Rey JA. Molecular analysis of chromosome 1 abnormalities in human gliomas reveals frequent loss of 1p in oligodendrogliomas. Int J Cancer 1994;57:172e5. [2] Reifenberger J, Reifenberger G, Liu L, James CD, Wechsler W, Collins VP. Molecular genetic analysis of oligodendroglial tumors shows preferential allelic deletions on 19q and 1p. Am J Pathol 1994;145:1175e90. [3] Bello MJ, Leone PE, Vaquero J, de Campos JM, Kusak ME, Sarasa JL, Pestana A, Rey JA. Allelic loss at 1p and 19q frequently occurs in association and may represent early oncogenic events in oligodendroglial tumors. Int J Cancer 1995;64:207e10. [4] Cairncross JG, Ueki K, Zlatescu C, Lisle DK, Finkelstein DM, Hammond RR, Silver JS, Stark PC, Macdonald DR, Ino Y, Ramsay DA, Louis DN. Specic genetic predictors of chemotherapeutic response and survival in patients with anaplastic oligodendroglioma. J Natl Cancer Inst 1998;90:1473e9. [5] Bello MJ, de Campos JM, Vaquero J, Ruiz-Barnes P, Kusak ME, Sarasa JL, Rey JA. hRAD54 gene and 1p high-resolution deletionmapping analyses in oligodendrogliomas. Cancer Genet Cytogenet 2000;116:142e7. [6] Alonso ME, Bello MJ, Gonzalez-Gomez P, Lomas J, de Campos JM, Kusak ME, Sarasa JL, Isla A, Rey JA. Mutation analysis of the p73 gene in nonastrocytic brain tumors. Br J Cancer 2001;85:204e8. [7] Barbashina V, Salazar P, Holland EC, Rosenblum MK, Ladanyi M. Allelic losses at 1p36 and 19q13 in gliomas: correlation with histologic classication, denition of a 150-kb minimal deletion region on 1p36, and evaluation of CAMTA1 as a candidate tumor suppressor gene. Clin Cancer Res 2005;11:1119e28. [8] Tews B, Felsberg J, Hartmann C, Kunitz A, Hahn M, Toedt G, Neben K, Hummerich L, von Deimling A, Reifenberger G, Lichter P. Identication of novel oligodendroglioma-associated tumor suppressor genes in 1p36 and 19q13 using microarray-based expression proling. Int J Cancer 2006;119:792e800. [9] Alonso ME, Bello MJ, Arjona D, Gonzalez-Gomez P, Aminoso C, Lopez-Marin I, de Campos JM, Isla A, Vaquero J, Gutierrez M, Sarasa JL, Rey JA. Mutational study of the 1p located gene p18ink4c, Patched-2, RIZ1 and KIF1B in oligodendrogliomas. Oncology Rep 2005;13:539e42. [10] Pohl U, Cairncross JG, Louis DN. Homozygous deletion of the CDKN2C/p18ink4 gene on the short arm of chromosome 1 in anaplastic oligodendrogliomas. Brain Pathol 1999;9:639e43. [11] Braganca J, Swingler T, Marques FI, Jones T, Eloranta JJ, Hurst HC, Shioda T, Bhattacharya S. Human CREB-binding protein/p300-interacting transactivator with ED-rich tail (CITED) 4, a new member of the CITED family, functions as a co-activator for transcription for AP-2. J Biol Chem 2002;277:8559e65. [12] Yahata T, Takedatsu H, Dunwoodie S, Braganca J, Swingler T, Withington S, Hur J, Coser K, Isselbacher K, Bhattacharya S, Shioda T. Cloning of mouse cited4, a member of the CITED family p300/CBP-binding transcriptional co-activator: induced expression in mammary epithelial cells. Genomics 2002;80:601e13.

10

170

11

173

Unreported

197

epigenetic alteration is frequent in astrocytic tumors, and aberrant hypermethylation of several tumor-related genes has been described in astrocytic and oligodendroglial tumors [18e20]. Preliminary data on CpG island CITED4 promoter methylation status in glioblastomas showed the epigenetic change in 2/14 glioblastomas studied (unpublished data) and both cases also presented loss of heterozygosity at 1p. By means of multiplex ligationedependent probe amplication (MLPA) analysis performed as described [21], the series reported here displayed deletions at 1p in 7/24 tumors. Partial loss at 1p36, 1p34, and 1p13.2 presented in one case each, and two additional tumors displayed complete 1p loss and an intact 19q. Concurrent losses at 1p/19q occurred in two samples and one glioblastoma displayed 19q loss and normal 1p allelic constitution. Thus, the data we obtained suggest that genetic (and perhaps epigenetic) mechanisms may contribute to the inactivation of CITED4 in a small fraction of glioblastomas, as proposed for oligodendroglial tumors [16].

Acknowledgements This study was supported by Fondo de Investigaciones Sanitarias, Ministerio de Sanidad (grant PI-05-0829). Carmen Franco-Hernandez is supported by a grant from Fun dacion para la Investigacion Biomedica del Hospital Universitario La Paz. Miguel Torres-Martn Carmen Franco-Hernandez Victor Martinez-Glez Jose M. de Campos

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Letter to the editor / Cancer Genetics and Cytogenetics 185 (2008) 114e116 neoplasms of the nervous system. Cancer Genet Cytogenet 1995; 83:160e4. Gonzalez-Gomez P, Bello MJ, Arjona D, Lomas J, Alonso ME, de Campos JM, Vaquero J, Isla A, Gutierrez M, Rey JA. Promoter hypermethylation of multiple genes in astrocytic gliomas. Int J Oncology 2003;22:601e8. Dong S-M, Pang JC-S, Poon W-S, Hu J, Yo K-F, Chang AR, Ng H-K. Concurrent methylation of multiple genes is associated with grade of oligodendroglial tumors. J Neuropathol Exp Neurol 2001;101: 185e9. Alonso ME, Bello MJ, Gonzalez-Gomez P, Arjona D, Lomas J, de Campos JM, Isla A, Sarasa JL, Rey JA. Aberrant promoter methylation of multiple genes in oligodendrogliomas and ependymomas. Cancer Genet Cytogenet 2003;144:134e42. Martinez-Glez V, Franco-Hernandez C, Lomas J, Pena-Granero C, de Campos JM, Isla A, Rey JA. Multiplex ligation-dependent probe amplication (MLPA) screening in meningioma. Cancer Genet Cytogenet 2007;173:170e2.

[13] Fox S, Braganca J, Turley H, Campo L, Han C, Gatter K, Bhattachary S, Harris A. CITED4 inhibits hypoxi-activated transcription in cancer cells, and its cytoplasmic location in breast cancer is associated with elevated expression of tumor cell hypoxia-inducible factor 1a. Cancer Res 2004;64:6075e81. [14] Yun W, Giordano A. Acetyltransferase machinery conserved in p300/CBP-family proteins. Oncogene 2002;21:2253e60. [15] Ogryzko V, Louis S, Russanova V, Howard B, Nakatani Y. The transcriptional co-activators p300 and CBP are histone acetyltransferases. Cell 1996;87:953e9. [16] Tews B, Roering P, Hartmann C, Hahn M, Felsberg J, Blaschke B, Sabel M, Kunitz A, Toedt G, Noben K, Benner A, von Deimling A, Reifenberger G, Lichter P. Hypermethylation and transcriptional downregulation of the CITED4 gene at 1p34.2 in oligodendroglial tumours with allelic losses on 1p and 19q. Oncogene 2007;26:5010e6. [17] Bello MJ, Leone PE, Nebreda P, de Campos JM, Kusak ME, Vaquero J, Sarasa JL, Garcia-Miguel P, Queizan A, Hernandez Moneo JL, Pestana A, Rey JA. Allelic status of chromosome 1 in

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