Limits...
Overexpression of ribosomal RNA in prostate cancer is common but not linked to rDNA promoter hypomethylation.

Uemura M, Zheng Q, Koh CM, Nelson WG, Yegnasubramanian S, De Marzo AM - Oncogene (2011)

Bottom Line: Further, as a surrogate for nucleolar size and number, we examined the expression of fibrillarin, which did not correlate with rRNA levels.We conclude that rRNA levels are increased in human prostate cancer, but that hypomethylation of the rDNA promoter does not explain this increase, nor does hypomethylation explain alterations in nucleolar number and structure in prostate cancer cells.Rather, rRNA levels and nucleolar size and number relate more closely to MYC overexpression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

ABSTRACT
Alterations in nucleoli, including increased numbers, increased size, altered architecture and increased function are hallmarks of prostate cancer cells. The mechanisms that result in increased nucleolar size, number and function in prostate cancer have not been fully elucidated. The nucleolus is formed around repeats of a transcriptional unit encoding a 45S ribosomal RNA (rRNA) precursor that is then processed to yield the mature 18S, 5.8S and 28S RNA species. Although it has been generally accepted that tumor cells overexpress rRNA species, this has not been examined in clinical prostate cancer. We find that indeed levels of the 45S rRNA, 28S, 18S and 5.8S are overexpressed in the majority of human primary prostate cancer specimens as compared with matched benign tissues. One mechanism that can alter nucleolar function and structure in cancer cells is hypomethylation of CpG dinucleotides of the upstream rDNA promoter region. However, this mechanism has not been examined in prostate cancer. To determine whether rRNA overexpression could be explained by hypomethylation of these CpG sites, we also evaluated the DNA methylation status of the rDNA promoter in prostate cancer cell lines and the clinical specimens. Bisulfite sequencing of genomic DNA revealed two roughly equal populations of loci in cell lines consisting of those that contained densely methylated deoxycytidine residues within CpGs and those that were largely unmethylated. All clinical specimens also contained two populations with no marked changes in methylation of this region in cancer as compared with normal. We recently reported that MYC can regulate rRNA levels in human prostate cancer; here we show that MYC mRNA levels are correlated with 45S, 18S and 5.8S rRNA levels. Further, as a surrogate for nucleolar size and number, we examined the expression of fibrillarin, which did not correlate with rRNA levels. We conclude that rRNA levels are increased in human prostate cancer, but that hypomethylation of the rDNA promoter does not explain this increase, nor does hypomethylation explain alterations in nucleolar number and structure in prostate cancer cells. Rather, rRNA levels and nucleolar size and number relate more closely to MYC overexpression.

Show MeSH

Related in: MedlinePlus

Methylation status of the rRNA promoter in the parental colorectal carcinoma cells HCT116 and its derivative knockout cell lines. The methylation status of each CpG dinucleotide spanning −158 to +29 bp of human rRNA promoter in HCT116 colorectal carcinoma cells and derivative lines with targeted disruption of DNMT1, DNMT3b or both genes. The rRNA promoter region was amplified from bisulfite-treated genomic DNA and cloned. The 16–19 randomly selected clones from each sample were subjected to automated sequencing. Each row represents the sequence of an individual clone, whereas each column depicts the position of the CpG. The filled and open circles denote methylated and unmethylated CpGs, respectively. The top row shows a heat map of methylated CpGs representing the frequency of methylation in each of the individual clones shown below. The degree of hypermethylation (NIM) at three CpG islands was assessed using quantitative real-time methylation-specific PCR and was normalized to the amount of bisulfite-converted input as described in ‘Materials and methods.'
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3298623&req=5

fig3: Methylation status of the rRNA promoter in the parental colorectal carcinoma cells HCT116 and its derivative knockout cell lines. The methylation status of each CpG dinucleotide spanning −158 to +29 bp of human rRNA promoter in HCT116 colorectal carcinoma cells and derivative lines with targeted disruption of DNMT1, DNMT3b or both genes. The rRNA promoter region was amplified from bisulfite-treated genomic DNA and cloned. The 16–19 randomly selected clones from each sample were subjected to automated sequencing. Each row represents the sequence of an individual clone, whereas each column depicts the position of the CpG. The filled and open circles denote methylated and unmethylated CpGs, respectively. The top row shows a heat map of methylated CpGs representing the frequency of methylation in each of the individual clones shown below. The degree of hypermethylation (NIM) at three CpG islands was assessed using quantitative real-time methylation-specific PCR and was normalized to the amount of bisulfite-converted input as described in ‘Materials and methods.'

Mentions: As a control to determine the ability of our bisulfite-sequencing approach to detect methylation changes in the rDNA promoter region, we first compared the methylation status in a series of isogenic colo-rectal carcinoma cell lines that contain either wild-type or disrupted versions of DNMT1, DMNT3b or both. A previous study found that targeted disruption of both DNMT1 and DNMT3b resulted in a total lack of methylation in this region (Gagnon-Kugler et al., 2009). Figure 3 shows results of bisulfite sequencing of the rDNA promoter in the colon cancer cell line HCT116 wild-type (HCT116_WT) or knockout mutants that lack either DNMT1 (HCT116_KO1), DNMT3b (HCT116_KO3b) or both genes (HCT116_DKO). In HCT116_WT, as expected there were two populations of alleles, one densely methylated and the other unmethylated. In HCT116_KO1 cells, there was a reduction in methylation, whereas in HCT116_KO3b cells there was less of a change overall, although the fraction of unmethylated alleles was somewhat increased. In HCT116_DKO cells there was a complete loss of methylation.


Overexpression of ribosomal RNA in prostate cancer is common but not linked to rDNA promoter hypomethylation.

Uemura M, Zheng Q, Koh CM, Nelson WG, Yegnasubramanian S, De Marzo AM - Oncogene (2011)

Methylation status of the rRNA promoter in the parental colorectal carcinoma cells HCT116 and its derivative knockout cell lines. The methylation status of each CpG dinucleotide spanning −158 to +29 bp of human rRNA promoter in HCT116 colorectal carcinoma cells and derivative lines with targeted disruption of DNMT1, DNMT3b or both genes. The rRNA promoter region was amplified from bisulfite-treated genomic DNA and cloned. The 16–19 randomly selected clones from each sample were subjected to automated sequencing. Each row represents the sequence of an individual clone, whereas each column depicts the position of the CpG. The filled and open circles denote methylated and unmethylated CpGs, respectively. The top row shows a heat map of methylated CpGs representing the frequency of methylation in each of the individual clones shown below. The degree of hypermethylation (NIM) at three CpG islands was assessed using quantitative real-time methylation-specific PCR and was normalized to the amount of bisulfite-converted input as described in ‘Materials and methods.'
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298623&req=5

fig3: Methylation status of the rRNA promoter in the parental colorectal carcinoma cells HCT116 and its derivative knockout cell lines. The methylation status of each CpG dinucleotide spanning −158 to +29 bp of human rRNA promoter in HCT116 colorectal carcinoma cells and derivative lines with targeted disruption of DNMT1, DNMT3b or both genes. The rRNA promoter region was amplified from bisulfite-treated genomic DNA and cloned. The 16–19 randomly selected clones from each sample were subjected to automated sequencing. Each row represents the sequence of an individual clone, whereas each column depicts the position of the CpG. The filled and open circles denote methylated and unmethylated CpGs, respectively. The top row shows a heat map of methylated CpGs representing the frequency of methylation in each of the individual clones shown below. The degree of hypermethylation (NIM) at three CpG islands was assessed using quantitative real-time methylation-specific PCR and was normalized to the amount of bisulfite-converted input as described in ‘Materials and methods.'
Mentions: As a control to determine the ability of our bisulfite-sequencing approach to detect methylation changes in the rDNA promoter region, we first compared the methylation status in a series of isogenic colo-rectal carcinoma cell lines that contain either wild-type or disrupted versions of DNMT1, DMNT3b or both. A previous study found that targeted disruption of both DNMT1 and DNMT3b resulted in a total lack of methylation in this region (Gagnon-Kugler et al., 2009). Figure 3 shows results of bisulfite sequencing of the rDNA promoter in the colon cancer cell line HCT116 wild-type (HCT116_WT) or knockout mutants that lack either DNMT1 (HCT116_KO1), DNMT3b (HCT116_KO3b) or both genes (HCT116_DKO). In HCT116_WT, as expected there were two populations of alleles, one densely methylated and the other unmethylated. In HCT116_KO1 cells, there was a reduction in methylation, whereas in HCT116_KO3b cells there was less of a change overall, although the fraction of unmethylated alleles was somewhat increased. In HCT116_DKO cells there was a complete loss of methylation.

Bottom Line: Further, as a surrogate for nucleolar size and number, we examined the expression of fibrillarin, which did not correlate with rRNA levels.We conclude that rRNA levels are increased in human prostate cancer, but that hypomethylation of the rDNA promoter does not explain this increase, nor does hypomethylation explain alterations in nucleolar number and structure in prostate cancer cells.Rather, rRNA levels and nucleolar size and number relate more closely to MYC overexpression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

ABSTRACT
Alterations in nucleoli, including increased numbers, increased size, altered architecture and increased function are hallmarks of prostate cancer cells. The mechanisms that result in increased nucleolar size, number and function in prostate cancer have not been fully elucidated. The nucleolus is formed around repeats of a transcriptional unit encoding a 45S ribosomal RNA (rRNA) precursor that is then processed to yield the mature 18S, 5.8S and 28S RNA species. Although it has been generally accepted that tumor cells overexpress rRNA species, this has not been examined in clinical prostate cancer. We find that indeed levels of the 45S rRNA, 28S, 18S and 5.8S are overexpressed in the majority of human primary prostate cancer specimens as compared with matched benign tissues. One mechanism that can alter nucleolar function and structure in cancer cells is hypomethylation of CpG dinucleotides of the upstream rDNA promoter region. However, this mechanism has not been examined in prostate cancer. To determine whether rRNA overexpression could be explained by hypomethylation of these CpG sites, we also evaluated the DNA methylation status of the rDNA promoter in prostate cancer cell lines and the clinical specimens. Bisulfite sequencing of genomic DNA revealed two roughly equal populations of loci in cell lines consisting of those that contained densely methylated deoxycytidine residues within CpGs and those that were largely unmethylated. All clinical specimens also contained two populations with no marked changes in methylation of this region in cancer as compared with normal. We recently reported that MYC can regulate rRNA levels in human prostate cancer; here we show that MYC mRNA levels are correlated with 45S, 18S and 5.8S rRNA levels. Further, as a surrogate for nucleolar size and number, we examined the expression of fibrillarin, which did not correlate with rRNA levels. We conclude that rRNA levels are increased in human prostate cancer, but that hypomethylation of the rDNA promoter does not explain this increase, nor does hypomethylation explain alterations in nucleolar number and structure in prostate cancer cells. Rather, rRNA levels and nucleolar size and number relate more closely to MYC overexpression.

Show MeSH
Related in: MedlinePlus