Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of CHD5 expression on tumor growth in neuroblastoma cell lines. NLF, IMR5, SK-N-SH, and SK-N-FI cells (107 each) stably transfected with plasmids containing CHD5- or CHD5-AS were injected into the flanks of nu/nu mice (n = 10 mice per group), and tumor growth was measured as volume weekly over 5 weeks. A) NLF CHD5-AS vs NLF CHD5, P = .002. B) IMR5 CHD5-AS vs IMR5 CHD5, P = .01. C) SK-N-SH CHD5-AS vs SK-N-SH CHD5, P = .07. D) SK-N-FI CHD5 vs SK-N-FI CHD5-AS, P = .26. Means and 95% confidence intervals (error bars) are shown. All P values (two-sided, comparisons at the 5-week time points) were calculated using a two-sample Student t test. Data are representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2483574&req=5

fig3: Effect of CHD5 expression on tumor growth in neuroblastoma cell lines. NLF, IMR5, SK-N-SH, and SK-N-FI cells (107 each) stably transfected with plasmids containing CHD5- or CHD5-AS were injected into the flanks of nu/nu mice (n = 10 mice per group), and tumor growth was measured as volume weekly over 5 weeks. A) NLF CHD5-AS vs NLF CHD5, P = .002. B) IMR5 CHD5-AS vs IMR5 CHD5, P = .01. C) SK-N-SH CHD5-AS vs SK-N-SH CHD5, P = .07. D) SK-N-FI CHD5 vs SK-N-FI CHD5-AS, P = .26. Means and 95% confidence intervals (error bars) are shown. All P values (two-sided, comparisons at the 5-week time points) were calculated using a two-sample Student t test. Data are representative of two independent experiments.

Mentions: Next, we injected neuroblastoma cells stably expressing CHD5 or CHD5-AS into the flanks of immunosuppressed nu/nu mice. Tumors became palpable within 1–2 weeks, but there was dramatic growth inhibition of CHD5-expressing clones for both NLF and IMR5 neuroblastoma cell lines compared with parental cell lines or CHD5-AS controls (Figure 3, A and B). The mean tumor volume for NLF-CHD5-AS at 5 weeks after injection was 1.65 cm3 (CI = 0.83 to 2.46 cm3), compared with 0.36 cm3 (CI = 0.17 to 0.44 cm3) for NLF-CHD5 (P = .002). The mean tumor size for IMR5-CHD5-AS at 5 weeks after injection was 1.15 cm3 (CI = 0.43 to 1.87 cm3), compared with 0.28 cm3 (CI = 0.18 to 0.38 cm3) for IMR5-CHD5 (P = .01). Both studies were repeated with similar results (data not shown). There was no statistically significant difference in growth rates between CHD5- and CHD5-AS–transfected SK-N-SH and SK-N-FI tumors (Figure 3, C and D), again demonstrating the specificity of tumor growth suppression for neuroblastoma 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 CHD5 expression on tumor growth in neuroblastoma cell lines. NLF, IMR5, SK-N-SH, and SK-N-FI cells (107 each) stably transfected with plasmids containing CHD5- or CHD5-AS were injected into the flanks of nu/nu mice (n = 10 mice per group), and tumor growth was measured as volume weekly over 5 weeks. A) NLF CHD5-AS vs NLF CHD5, P = .002. B) IMR5 CHD5-AS vs IMR5 CHD5, P = .01. C) SK-N-SH CHD5-AS vs SK-N-SH CHD5, P = .07. D) SK-N-FI CHD5 vs SK-N-FI CHD5-AS, P = .26. Means and 95% confidence intervals (error bars) are shown. All P values (two-sided, comparisons at the 5-week time points) were calculated using a two-sample Student t test. Data are representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Effect of CHD5 expression on tumor growth in neuroblastoma cell lines. NLF, IMR5, SK-N-SH, and SK-N-FI cells (107 each) stably transfected with plasmids containing CHD5- or CHD5-AS were injected into the flanks of nu/nu mice (n = 10 mice per group), and tumor growth was measured as volume weekly over 5 weeks. A) NLF CHD5-AS vs NLF CHD5, P = .002. B) IMR5 CHD5-AS vs IMR5 CHD5, P = .01. C) SK-N-SH CHD5-AS vs SK-N-SH CHD5, P = .07. D) SK-N-FI CHD5 vs SK-N-FI CHD5-AS, P = .26. Means and 95% confidence intervals (error bars) are shown. All P values (two-sided, comparisons at the 5-week time points) were calculated using a two-sample Student t test. Data are representative of two independent experiments.
Mentions: Next, we injected neuroblastoma cells stably expressing CHD5 or CHD5-AS into the flanks of immunosuppressed nu/nu mice. Tumors became palpable within 1–2 weeks, but there was dramatic growth inhibition of CHD5-expressing clones for both NLF and IMR5 neuroblastoma cell lines compared with parental cell lines or CHD5-AS controls (Figure 3, A and B). The mean tumor volume for NLF-CHD5-AS at 5 weeks after injection was 1.65 cm3 (CI = 0.83 to 2.46 cm3), compared with 0.36 cm3 (CI = 0.17 to 0.44 cm3) for NLF-CHD5 (P = .002). The mean tumor size for IMR5-CHD5-AS at 5 weeks after injection was 1.15 cm3 (CI = 0.43 to 1.87 cm3), compared with 0.28 cm3 (CI = 0.18 to 0.38 cm3) for IMR5-CHD5 (P = .01). Both studies were repeated with similar results (data not shown). There was no statistically significant difference in growth rates between CHD5- and CHD5-AS–transfected SK-N-SH and SK-N-FI tumors (Figure 3, C and D), again demonstrating the specificity of tumor growth suppression for neuroblastoma 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