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Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells.

Mathews LA, Hurt EM, Zhang X, Farrar WL - Mol. Cancer (2010)

Bottom Line: The non-receptor tyrosine kinase BMX and transcription factor SOX1 were found to play a significant role in invasion.Cells which have decreased levels of the targets BMX and SOX1 also display loss of STAT3 activity.Using this method we can begin to understand which genes are epigenetically regulated in the invasive population compared to the bulk tumor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Stem Cell Section, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA.

ABSTRACT

Background: Recently, much attention has been focused on gaining a better understanding of the different populations of cells within a tumor and their contribution to cancer progression. One of the most commonly used methods to isolate a more aggressive sub-population of cells utilizes cell sorting based on expression of certain cell adhesion molecules. A recently established method we developed is to isolate these more aggressive cells based on their properties of increased invasive ability. These more invasive cells have been previously characterized as tumor initiating cells (TICs) that have a stem-like genomic signature and express a number of stem cell genes including Oct3/4 and Nanog and are more tumorigenic compared to their 'non-invasive' counterpart. They also have a profile reminiscent of cells undergoing a classic pattern of epithelial to mesenchymal transition or EMT. Using this model of invasion, we sought to investigate which genes are under epigenetic control in this rare population of cells. Epigenetic modifications, specifically DNA methylation, are key events regulating the process of normal human development. To determine the specific methylation pattern in these invasive prostate cells, and if any developmental genes were being differentially regulated, we analyzed differences in global CpG promoter methylation.

Results: Differentially methylated genes were determined and select genes were chosen for additional analyses. The non-receptor tyrosine kinase BMX and transcription factor SOX1 were found to play a significant role in invasion. Ingenuity pathway analysis revealed the methylated gene list frequently displayed genes from the IL-6/STAT3 pathway. Cells which have decreased levels of the targets BMX and SOX1 also display loss of STAT3 activity. Finally, using Oncomine, it was determined that more aggressive metastatic prostate cancers in humans also have higher levels of both Stat3 and Sox1.

Conclusions: Using this method we can begin to understand which genes are epigenetically regulated in the invasive population compared to the bulk tumor cells. These aggressive sub-populations of cells may be linked to the cancer stem cell hypothesis, making their patterns of epigenetic regulation very attractive for biomarker analysis.

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Related in: MedlinePlus

Validation of methylated targets in LNcaP and DU145 cells. A) DNA was extracted using the DNeasy kit and total of 1 μg from parental (total) LNCaP and DU145 cells was bisulfite modified using the EpiTect Bisulfite kit from Qiagen. MS-PCR was performed using Platinum Taq Polymerase (Invitrogen) and 200 ng of either genomic of bisulfite treated DNA was used. The samples were visualized using a 1% agarose gel and ethidium bromide. Both Sox1 and Bmx are methylated in the LNCaP and DU145 cell lines. B) Total RNA was isolated using TRIzol and qRT-PCR analysis was performed using a StepOne Real-time PCR machine with TaqMan Gene Expression Assay reagents and probes. Isolation of DNA and cDNA from non-invasive and invasive cells was carried out as previously described in materials and methods. Relative fold induction of mRNA was compared between non-invasive and invasive cells using the Delta-Delta CT method of quantitation where the parental lines were set at 1.0 as the control, and 18S rRNA was used as a loading control. Increased levels of both Sox1 and Bmx are seen in invasive LNCaP and DU145 cells compared to the non-invasive and parental lines. Normal human prostate RNA was used as a control. A Two-way ANOVA with a Bonferroni post-test was performed to compare groups and * represents a p-value of < 0.05 comparing parental to non-invasive cells and ** comparing parental to invasive cells. C) Staining of invasive or non-invasive cells was performed directly on the Matrigel membrane. Cells were incubated with either anti-pBMX antibody or SOX1 overnight and goat anti-rabbit Alexa-488 was added for 1 hour. Membranes were mounted on glass slides with Vectashield containing DAPI and visualized with a Zeiss-510 L5 confocal microscope. Images were analyzed using the Zeiss LSM5 Image Browser (20×) and further prepared in Adobe Photoshop CS. Increased levels of pBMX and SOX1 are seen in invasive cells compared to the non-invasive cells on top of the membrane.
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Figure 3: Validation of methylated targets in LNcaP and DU145 cells. A) DNA was extracted using the DNeasy kit and total of 1 μg from parental (total) LNCaP and DU145 cells was bisulfite modified using the EpiTect Bisulfite kit from Qiagen. MS-PCR was performed using Platinum Taq Polymerase (Invitrogen) and 200 ng of either genomic of bisulfite treated DNA was used. The samples were visualized using a 1% agarose gel and ethidium bromide. Both Sox1 and Bmx are methylated in the LNCaP and DU145 cell lines. B) Total RNA was isolated using TRIzol and qRT-PCR analysis was performed using a StepOne Real-time PCR machine with TaqMan Gene Expression Assay reagents and probes. Isolation of DNA and cDNA from non-invasive and invasive cells was carried out as previously described in materials and methods. Relative fold induction of mRNA was compared between non-invasive and invasive cells using the Delta-Delta CT method of quantitation where the parental lines were set at 1.0 as the control, and 18S rRNA was used as a loading control. Increased levels of both Sox1 and Bmx are seen in invasive LNCaP and DU145 cells compared to the non-invasive and parental lines. Normal human prostate RNA was used as a control. A Two-way ANOVA with a Bonferroni post-test was performed to compare groups and * represents a p-value of < 0.05 comparing parental to non-invasive cells and ** comparing parental to invasive cells. C) Staining of invasive or non-invasive cells was performed directly on the Matrigel membrane. Cells were incubated with either anti-pBMX antibody or SOX1 overnight and goat anti-rabbit Alexa-488 was added for 1 hour. Membranes were mounted on glass slides with Vectashield containing DAPI and visualized with a Zeiss-510 L5 confocal microscope. Images were analyzed using the Zeiss LSM5 Image Browser (20×) and further prepared in Adobe Photoshop CS. Increased levels of pBMX and SOX1 are seen in invasive cells compared to the non-invasive cells on top of the membrane.

Mentions: To verify the results from our methylation specific promoter tiling arrays, we performed methylation specific PCR (MS-PCR) where primers were designed around the probe sequences identified from the arrays. Both Bmx and Sox1 were found to be methylated in the parental (total) LNCaP and DU145 cell lines (Figure 3A), representing the non-invasive phenotype. To determine if this pattern of methylation correlated with the level of gene expression, real time quantitative PCR (qRT-PCR) was performed. Significant differences in the expression of Bmx and Sox1 were seen when comparing the expression in non-invasive and invasive cell populations in both LNCaP and DU145 cell lines (Figure 3B) (Two-way ANOVA; *compares non-invasive to parental and ** compares invasive to parental, p < 0.05). To further validate the results, immunocytochemistry (ICC) was performed to analyze differences in protein expression between non-invasive and invasive cells. There is significantly higher expression of activated BMX and SOX1 in the invasive versus non-invasive cells (Figure 3C). Therefore, we validated the methylation and resultant decreased expression of BMX and SOX1 in the non-invasive cells.


Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells.

Mathews LA, Hurt EM, Zhang X, Farrar WL - Mol. Cancer (2010)

Validation of methylated targets in LNcaP and DU145 cells. A) DNA was extracted using the DNeasy kit and total of 1 μg from parental (total) LNCaP and DU145 cells was bisulfite modified using the EpiTect Bisulfite kit from Qiagen. MS-PCR was performed using Platinum Taq Polymerase (Invitrogen) and 200 ng of either genomic of bisulfite treated DNA was used. The samples were visualized using a 1% agarose gel and ethidium bromide. Both Sox1 and Bmx are methylated in the LNCaP and DU145 cell lines. B) Total RNA was isolated using TRIzol and qRT-PCR analysis was performed using a StepOne Real-time PCR machine with TaqMan Gene Expression Assay reagents and probes. Isolation of DNA and cDNA from non-invasive and invasive cells was carried out as previously described in materials and methods. Relative fold induction of mRNA was compared between non-invasive and invasive cells using the Delta-Delta CT method of quantitation where the parental lines were set at 1.0 as the control, and 18S rRNA was used as a loading control. Increased levels of both Sox1 and Bmx are seen in invasive LNCaP and DU145 cells compared to the non-invasive and parental lines. Normal human prostate RNA was used as a control. A Two-way ANOVA with a Bonferroni post-test was performed to compare groups and * represents a p-value of < 0.05 comparing parental to non-invasive cells and ** comparing parental to invasive cells. C) Staining of invasive or non-invasive cells was performed directly on the Matrigel membrane. Cells were incubated with either anti-pBMX antibody or SOX1 overnight and goat anti-rabbit Alexa-488 was added for 1 hour. Membranes were mounted on glass slides with Vectashield containing DAPI and visualized with a Zeiss-510 L5 confocal microscope. Images were analyzed using the Zeiss LSM5 Image Browser (20×) and further prepared in Adobe Photoshop CS. Increased levels of pBMX and SOX1 are seen in invasive cells compared to the non-invasive cells on top of the membrane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Validation of methylated targets in LNcaP and DU145 cells. A) DNA was extracted using the DNeasy kit and total of 1 μg from parental (total) LNCaP and DU145 cells was bisulfite modified using the EpiTect Bisulfite kit from Qiagen. MS-PCR was performed using Platinum Taq Polymerase (Invitrogen) and 200 ng of either genomic of bisulfite treated DNA was used. The samples were visualized using a 1% agarose gel and ethidium bromide. Both Sox1 and Bmx are methylated in the LNCaP and DU145 cell lines. B) Total RNA was isolated using TRIzol and qRT-PCR analysis was performed using a StepOne Real-time PCR machine with TaqMan Gene Expression Assay reagents and probes. Isolation of DNA and cDNA from non-invasive and invasive cells was carried out as previously described in materials and methods. Relative fold induction of mRNA was compared between non-invasive and invasive cells using the Delta-Delta CT method of quantitation where the parental lines were set at 1.0 as the control, and 18S rRNA was used as a loading control. Increased levels of both Sox1 and Bmx are seen in invasive LNCaP and DU145 cells compared to the non-invasive and parental lines. Normal human prostate RNA was used as a control. A Two-way ANOVA with a Bonferroni post-test was performed to compare groups and * represents a p-value of < 0.05 comparing parental to non-invasive cells and ** comparing parental to invasive cells. C) Staining of invasive or non-invasive cells was performed directly on the Matrigel membrane. Cells were incubated with either anti-pBMX antibody or SOX1 overnight and goat anti-rabbit Alexa-488 was added for 1 hour. Membranes were mounted on glass slides with Vectashield containing DAPI and visualized with a Zeiss-510 L5 confocal microscope. Images were analyzed using the Zeiss LSM5 Image Browser (20×) and further prepared in Adobe Photoshop CS. Increased levels of pBMX and SOX1 are seen in invasive cells compared to the non-invasive cells on top of the membrane.
Mentions: To verify the results from our methylation specific promoter tiling arrays, we performed methylation specific PCR (MS-PCR) where primers were designed around the probe sequences identified from the arrays. Both Bmx and Sox1 were found to be methylated in the parental (total) LNCaP and DU145 cell lines (Figure 3A), representing the non-invasive phenotype. To determine if this pattern of methylation correlated with the level of gene expression, real time quantitative PCR (qRT-PCR) was performed. Significant differences in the expression of Bmx and Sox1 were seen when comparing the expression in non-invasive and invasive cell populations in both LNCaP and DU145 cell lines (Figure 3B) (Two-way ANOVA; *compares non-invasive to parental and ** compares invasive to parental, p < 0.05). To further validate the results, immunocytochemistry (ICC) was performed to analyze differences in protein expression between non-invasive and invasive cells. There is significantly higher expression of activated BMX and SOX1 in the invasive versus non-invasive cells (Figure 3C). Therefore, we validated the methylation and resultant decreased expression of BMX and SOX1 in the non-invasive cells.

Bottom Line: The non-receptor tyrosine kinase BMX and transcription factor SOX1 were found to play a significant role in invasion.Cells which have decreased levels of the targets BMX and SOX1 also display loss of STAT3 activity.Using this method we can begin to understand which genes are epigenetically regulated in the invasive population compared to the bulk tumor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Stem Cell Section, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA.

ABSTRACT

Background: Recently, much attention has been focused on gaining a better understanding of the different populations of cells within a tumor and their contribution to cancer progression. One of the most commonly used methods to isolate a more aggressive sub-population of cells utilizes cell sorting based on expression of certain cell adhesion molecules. A recently established method we developed is to isolate these more aggressive cells based on their properties of increased invasive ability. These more invasive cells have been previously characterized as tumor initiating cells (TICs) that have a stem-like genomic signature and express a number of stem cell genes including Oct3/4 and Nanog and are more tumorigenic compared to their 'non-invasive' counterpart. They also have a profile reminiscent of cells undergoing a classic pattern of epithelial to mesenchymal transition or EMT. Using this model of invasion, we sought to investigate which genes are under epigenetic control in this rare population of cells. Epigenetic modifications, specifically DNA methylation, are key events regulating the process of normal human development. To determine the specific methylation pattern in these invasive prostate cells, and if any developmental genes were being differentially regulated, we analyzed differences in global CpG promoter methylation.

Results: Differentially methylated genes were determined and select genes were chosen for additional analyses. The non-receptor tyrosine kinase BMX and transcription factor SOX1 were found to play a significant role in invasion. Ingenuity pathway analysis revealed the methylated gene list frequently displayed genes from the IL-6/STAT3 pathway. Cells which have decreased levels of the targets BMX and SOX1 also display loss of STAT3 activity. Finally, using Oncomine, it was determined that more aggressive metastatic prostate cancers in humans also have higher levels of both Stat3 and Sox1.

Conclusions: Using this method we can begin to understand which genes are epigenetically regulated in the invasive population compared to the bulk tumor cells. These aggressive sub-populations of cells may be linked to the cancer stem cell hypothesis, making their patterns of epigenetic regulation very attractive for biomarker analysis.

Show MeSH
Related in: MedlinePlus