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CHD5, a tumor suppressor gene deleted from 1p36.31 in neuroblastomas.

Fujita T, Igarashi J, Okawa ER, Gotoh T, Manne J, Kolla V, Kim J, Zhao H, Pawel BR, London WB, Maris JM, White PS, Brodeur GM - J. Natl. Cancer Inst. (2008)

Bottom Line: We also analyzed the association of CDH5 expression with outcomes of 99 neuroblastoma patients.The CHD5 promoter was highly methylated in NLF and IMR5 lines, and CHD5 expression increased after treatment with 5-aza-2-deoxycytidine.High CHD5 expression was strongly associated with favorable event-free and overall survival (P < .001), even after correction for MYCN amplification and 1p deletion (P = .027).

View Article: PubMed Central - PubMed

Affiliation: Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, PA 19104-4318, USA.

ABSTRACT

Background: Neuroblastomas are characterized by hemizygous 1p deletions, suggesting that a tumor suppressor gene resides in this region. We previously mapped the smallest region of consistent deletion to a 2-Mb region of 1p36.31 that encodes 23 genes. Based on mutation analysis, expression pattern, and putative function, we identified CHD5 as the best tumor suppressor gene candidate.

Methods: We determined the methylation status of the CHD5 gene promoter in NLF and IMR5 (with 1p deletion) and SK-N-SH and SK-N-FI neuroblastoma cell lines using methylation-specific sequencing and measured CHD5 mRNA expression by reverse transcription polymerase chain reaction in cells treated with or without 5-aza-2-deoxycytidine, an inhibitor of DNA methylation. We transfected the cells with CHD5 and antisense (AS) CHD5 DNA to assess the effect of CHD5 overexpression and suppression, respectively, on colony formation in soft agar and growth of xenograft tumors in athymic mice. We also analyzed the association of CDH5 expression with outcomes of 99 neuroblastoma patients. Statistical tests were two-sided.

Results: CHD5 expression was very low or absent in neuroblastoma cell lines. The CHD5 promoter was highly methylated in NLF and IMR5 lines, and CHD5 expression increased after treatment with 5-aza-2-deoxycytidine. Clonogenicity and tumor growth were abrogated in NLF and IMR5 cells overexpressing CHD5 compared with antisense CHD5 (clonogenicity: mean no. of colonies per plate, NLF-CHD5, 43 colonies, 95% confidence interval [CI] = 35 to 51 colonies, vs NLF-CHD5-AS, 74 colonies, 95% CI = 62 to 86 colonies, P < .001; IMR5-CHD5, 11 colonies, 95% CI = 2 to 20 colonies, vs IMR5-CHD5-AS, 39 colonies, 95% CI = 17 to 60 colonies, P = .01; tumor growth, n = 10 mice per group: mean tumor size at 5 weeks, NLF-CHD5, 0.36 cm(3), 95% CI = 0.17 to 0.44 cm(3), vs NLF-CHD5-AS, 1.65 cm(3), 95% CI = 0.83 to 2.46 cm(3), P = .002; IMR5-CHD5, 0.28 cm(3), 95% CI = 0.18 to 0.38 cm(3), vs IMR5-CHD5-AS, 1.15 cm(3), 95% CI = 0.43 to 1.87 cm(3); P = .01). High CHD5 expression was strongly associated with favorable event-free and overall survival (P < .001), even after correction for MYCN amplification and 1p deletion (P = .027).

Conclusions: CHD5 is the strongest candidate tumor suppressor gene that is deleted from 1p36.31 in neuroblastomas, and inactivation of the second allele may occur by an epigenetic mechanism.

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Effect of altered CHD5 expression on clonogenicity in neuroblastoma cell lines. Plasmids containing CHD5 or antisense CHD5 (CHD5-AS) were stably transfected into NLF and IMR5 neuroblastoma cell lines, both of which have hemizygous 1p deletion and MYCN amplification, and into SK-N-SH and SK-N-FI neuroblastoma cell lines, which have neither. Transfected cells were plated on soft agar, and colonies were counted 3 weeks later. A) NLF cells. CHD5 vs CHD5-AS, P < .001. B) IMR5 cells. CHD5 vs CHD5-AS, P = .001. C) SK-N-SH cells. CHD5 vs CHD5-AS, P = .16. D) SK-N-FI cells. CHD5 vs CHD5-AS, P = .10. Means and 95% confidence intervals (error bars) are shown. P values (two-sided) were calculated using the Student t test. Data are representative of three independent experiments performed in quadruplicate.
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fig2: Effect of altered CHD5 expression on clonogenicity in neuroblastoma cell lines. Plasmids containing CHD5 or antisense CHD5 (CHD5-AS) were stably transfected into NLF and IMR5 neuroblastoma cell lines, both of which have hemizygous 1p deletion and MYCN amplification, and into SK-N-SH and SK-N-FI neuroblastoma cell lines, which have neither. Transfected cells were plated on soft agar, and colonies were counted 3 weeks later. A) NLF cells. CHD5 vs CHD5-AS, P < .001. B) IMR5 cells. CHD5 vs CHD5-AS, P = .001. C) SK-N-SH cells. CHD5 vs CHD5-AS, P = .16. D) SK-N-FI cells. CHD5 vs CHD5-AS, P = .10. Means and 95% confidence intervals (error bars) are shown. P values (two-sided) were calculated using the Student t test. Data are representative of three independent experiments performed in quadruplicate.

Mentions: Next, we plated cells from CHD5-transfected and CHD5-AS–transfected lines into soft agar. After 3 weeks of culture, NLF-CHD5-AS clones formed statistically significantly more colonies (mean = 74 large colonies per plate, 95% confidence interval [CI] = 62 to 86 colonies) than NLF-CHD5 clones (mean = 43 colonies, 95% CI = 35 to 51 colonies (P < .001) (Figure 2, A). Similarly, the IMR5-CHD5-AS control cells formed more colonies (mean = 39 colonies, 95% CI = 17 to 60 colonies) than the CHD5-transfected cells (mean = 11 colonies, 95% CI = 2 to 20 colonies) (P = .01) (Figure 2, B). However, there was no difference between the number of colonies formed by CHD5-transfected and CHD5-AS–transfected clones of SK-N-SH (P = .16) and SK-N-FI (P = .1) (Figure 2, C and D). Thus, CHD5 expression caused a statistically significant reduction in colony formation in the CHD5-expressing clones relative to the CHD5-AS clones, but only in cell lines with 1p deletion.


CHD5, a tumor suppressor gene deleted from 1p36.31 in neuroblastomas.

Fujita T, Igarashi J, Okawa ER, Gotoh T, Manne J, Kolla V, Kim J, Zhao H, Pawel BR, London WB, Maris JM, White PS, Brodeur GM - J. Natl. Cancer Inst. (2008)

Effect of altered CHD5 expression on clonogenicity in neuroblastoma cell lines. Plasmids containing CHD5 or antisense CHD5 (CHD5-AS) were stably transfected into NLF and IMR5 neuroblastoma cell lines, both of which have hemizygous 1p deletion and MYCN amplification, and into SK-N-SH and SK-N-FI neuroblastoma cell lines, which have neither. Transfected cells were plated on soft agar, and colonies were counted 3 weeks later. A) NLF cells. CHD5 vs CHD5-AS, P < .001. B) IMR5 cells. CHD5 vs CHD5-AS, P = .001. C) SK-N-SH cells. CHD5 vs CHD5-AS, P = .16. D) SK-N-FI cells. CHD5 vs CHD5-AS, P = .10. Means and 95% confidence intervals (error bars) are shown. P values (two-sided) were calculated using the Student t test. Data are representative of three independent experiments performed in quadruplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483574&req=5

fig2: Effect of altered CHD5 expression on clonogenicity in neuroblastoma cell lines. Plasmids containing CHD5 or antisense CHD5 (CHD5-AS) were stably transfected into NLF and IMR5 neuroblastoma cell lines, both of which have hemizygous 1p deletion and MYCN amplification, and into SK-N-SH and SK-N-FI neuroblastoma cell lines, which have neither. Transfected cells were plated on soft agar, and colonies were counted 3 weeks later. A) NLF cells. CHD5 vs CHD5-AS, P < .001. B) IMR5 cells. CHD5 vs CHD5-AS, P = .001. C) SK-N-SH cells. CHD5 vs CHD5-AS, P = .16. D) SK-N-FI cells. CHD5 vs CHD5-AS, P = .10. Means and 95% confidence intervals (error bars) are shown. P values (two-sided) were calculated using the Student t test. Data are representative of three independent experiments performed in quadruplicate.
Mentions: Next, we plated cells from CHD5-transfected and CHD5-AS–transfected lines into soft agar. After 3 weeks of culture, NLF-CHD5-AS clones formed statistically significantly more colonies (mean = 74 large colonies per plate, 95% confidence interval [CI] = 62 to 86 colonies) than NLF-CHD5 clones (mean = 43 colonies, 95% CI = 35 to 51 colonies (P < .001) (Figure 2, A). Similarly, the IMR5-CHD5-AS control cells formed more colonies (mean = 39 colonies, 95% CI = 17 to 60 colonies) than the CHD5-transfected cells (mean = 11 colonies, 95% CI = 2 to 20 colonies) (P = .01) (Figure 2, B). However, there was no difference between the number of colonies formed by CHD5-transfected and CHD5-AS–transfected clones of SK-N-SH (P = .16) and SK-N-FI (P = .1) (Figure 2, C and D). Thus, CHD5 expression caused a statistically significant reduction in colony formation in the CHD5-expressing clones relative to the CHD5-AS clones, but only in cell lines with 1p deletion.

Bottom Line: We also analyzed the association of CDH5 expression with outcomes of 99 neuroblastoma patients.The CHD5 promoter was highly methylated in NLF and IMR5 lines, and CHD5 expression increased after treatment with 5-aza-2-deoxycytidine.High CHD5 expression was strongly associated with favorable event-free and overall survival (P < .001), even after correction for MYCN amplification and 1p deletion (P = .027).

View Article: PubMed Central - PubMed

Affiliation: Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, PA 19104-4318, USA.

ABSTRACT

Background: Neuroblastomas are characterized by hemizygous 1p deletions, suggesting that a tumor suppressor gene resides in this region. We previously mapped the smallest region of consistent deletion to a 2-Mb region of 1p36.31 that encodes 23 genes. Based on mutation analysis, expression pattern, and putative function, we identified CHD5 as the best tumor suppressor gene candidate.

Methods: We determined the methylation status of the CHD5 gene promoter in NLF and IMR5 (with 1p deletion) and SK-N-SH and SK-N-FI neuroblastoma cell lines using methylation-specific sequencing and measured CHD5 mRNA expression by reverse transcription polymerase chain reaction in cells treated with or without 5-aza-2-deoxycytidine, an inhibitor of DNA methylation. We transfected the cells with CHD5 and antisense (AS) CHD5 DNA to assess the effect of CHD5 overexpression and suppression, respectively, on colony formation in soft agar and growth of xenograft tumors in athymic mice. We also analyzed the association of CDH5 expression with outcomes of 99 neuroblastoma patients. Statistical tests were two-sided.

Results: CHD5 expression was very low or absent in neuroblastoma cell lines. The CHD5 promoter was highly methylated in NLF and IMR5 lines, and CHD5 expression increased after treatment with 5-aza-2-deoxycytidine. Clonogenicity and tumor growth were abrogated in NLF and IMR5 cells overexpressing CHD5 compared with antisense CHD5 (clonogenicity: mean no. of colonies per plate, NLF-CHD5, 43 colonies, 95% confidence interval [CI] = 35 to 51 colonies, vs NLF-CHD5-AS, 74 colonies, 95% CI = 62 to 86 colonies, P < .001; IMR5-CHD5, 11 colonies, 95% CI = 2 to 20 colonies, vs IMR5-CHD5-AS, 39 colonies, 95% CI = 17 to 60 colonies, P = .01; tumor growth, n = 10 mice per group: mean tumor size at 5 weeks, NLF-CHD5, 0.36 cm(3), 95% CI = 0.17 to 0.44 cm(3), vs NLF-CHD5-AS, 1.65 cm(3), 95% CI = 0.83 to 2.46 cm(3), P = .002; IMR5-CHD5, 0.28 cm(3), 95% CI = 0.18 to 0.38 cm(3), vs IMR5-CHD5-AS, 1.15 cm(3), 95% CI = 0.43 to 1.87 cm(3); P = .01). High CHD5 expression was strongly associated with favorable event-free and overall survival (P < .001), even after correction for MYCN amplification and 1p deletion (P = .027).

Conclusions: CHD5 is the strongest candidate tumor suppressor gene that is deleted from 1p36.31 in neuroblastomas, and inactivation of the second allele may occur by an epigenetic mechanism.

Show MeSH
Related in: MedlinePlus