Amongst the primary tumor samples, 20 27 samples showed low expression of PAX6 as compared to normal adult brain tissue. Interestingly, most of the astro cytic tumor samples showed low expression levels of PAX6 even though GLI1 was expressed at high levels. NKX2. 2 Silencing of GLI1 resulted in especially a decrease in NKX2. 2 by 50% in the Daoy cell line compared with scrambled siRNA transfected and untransfected cells. Expression of NKX2. 2 was low in 2 cell lines, high in only one and was not expressed in the remaining 3. Primary medulloblasto mas also showed a similar pattern of low expression of NKX2. 2 and this correlated with high expression levels of PAX6. Six astrocytic cell lines showed very low expression of NKX2. 2, and the remaining 2 cell lines did not express it at all, compared with expres sion levels in normal adult brain tissue.
Out of the 27 astrocytic tumor samples, 20 showed low expression of NKX2. 2 compared with normal adult brain tissue. Amongst the samples, low grade tumors did not express NKX2. 2. however, few of the high grade samples showed very high expression levels of NKX2. 2 and low expres sion of GLI1. Overall, most samples expressed NKX2. 2 at low levels while expressing GLI1 to a high degree. No correlation between GLI1 and Cyclin D2 protein expression GLI1 protein was expressed in most astrocytic cell lines with the exception of two. However, very low expression of Cyclin D2 protein was observed in the following seven cell lines U87MG, A172, LN405, SW1088, T98G, CCF STTG 1 and GOS 3. moreover, there was no expression of Cyclin D2 in one cell line.
Most of the primary tumor samples showed high expression levels of the GLI1 protein but these samples did not express Cyclin D2. Cyclin D2 and PTCH1 epigenetics We did not observe any changes in the pattern of Cyclin D2 expression upon treatment of the medulloblastoma cell lines with 5 Aza 2 deoxycytidine and TSA. How ever, treatment with these compounds resulted in the onset of Cyclin D2 expression, assessed by both RT PCR and qRT PCR in five astrocytoma cell lines that did not initially express Cyclin D2. The increase in Cyclin D2 mRNA in these cell lines was statistically significant. Amongst the 6 medulloblastoma cell lines assayed using the MCA Meth method, only PFSK 1 and SK PN DW showed a hemi methylation melting curve. However, hypermethylation was revealed in all cell lines by MSP.
Methylation asso ciated with low Cyclin D2 expression was evident only in the SK PN DW cell line. Drug_discovery In medulloblas toma samples, no methylation was detected by MCA Meth primers, even though some tumors expressed Cyclin D2 at low levels. However, MSP showed promoter hypermethylation which was asso ciated with the lack of mRNA expression or low expres sion in tumor samples. In astrocytomas, methylation analysis by MCA Meth and MSP PCR methods was performed on 8 cell lines.