The in vitro-in vivo
model described in the previous section represents different stages of cancer progression, MCF-10F being the normal stage; trMCF, the transformed stage; bsMCF, the invasive stage; and a more advance stage represented by caMCF (). These phenotypes correlated with gene dysregulation during the progression of the transformation. The highest number of dysregulated genes was observed in caMCF, being slightly lower in bsMCF, and lowest in trMCF. This order was consistent with the extent of chromosome aberrations (caMCF > bsMCF
> trMCF) that were studied using comparative genomic hybridization (CGH) (). For CGH, the DNA hybridization and digital image analysis were performed as previously described (Fernandez et al., 2005
). Gain or losses are progressive at the different stages of the in vitro-in vivo
model; there are few gains or losses in trMCF and, more in the invasive bsMCF and the tumorigenic caMCF cells (). Only small chromosome gains were observed in trMCF and most alterations were observed in bsMCF and caMCF cells (). Chromosomal amplifications were found in 1p36.12-pter, 5q21.1-qter in both bsMCF and caMCF. Losses of the complete chromosome 4 and 8p11.21–23.1 were found in bsMCF and caMCF cells (). In caMCF cells, additional losses were found in 3p12.1–14.1, 9p22.1-pter and 18q11.21-qter. Also a chromosomal amplification in 13q21.31-qter was found in caMCF using high density SNP arrays (Huang et al., 2007
). Functional profiling of dysregulated genes revealed progressive changes in the integrin signaling pathway, inhibition of apoptosis, acquisition of tumorigenic cell surface markers and epithelial-mesenchymal transition (EMT). In tumorigenic cells, the levels of E-cadherin, EMA, and various keratins were low and CD44E/CD24 were negative, whereas SNAI2, vimentin, S100A4, FN1, HRAS, TGFβ1, and CD44H were high.
Comparative genomic hybridization (CGH) analysis of the in vitro-in vivo model of cell transformation induced by E2
Global CpG island methylation at the different stages in the in vitro-in vivo
model of cell transformation was studied using restriction landmark genomic scanning (RLGS) (Fernandez et al., 2006b
). RLGS is based on DNA digestion with the restriction enzyme NotI which is methylation-sensitive because it is only able to cut unmethylated DNA (). The sites of differential methylation in trMCF, bsMCF and caMCF cells were compared with MCF-10F methylation profile. A total of thirty-eight genes were identified as hypermethylated and four genes were hypomethylated when compared trMCF, bsMCF and caMCF with the control MCF-10F cells (). The data revealed that the methylation pattern of different genes related to ductulogenesis and branching, estrogen metabolism, apoptosis and proliferation was altered (). This is the first demonstration that changes in DNA methylation are involved in the early and late stages of breast cancer progression indicating that from the thirty-eight hypermethylated genes, seventeen of them were potentially involved in the branching process: HOXA9, HOXB5, EPB49, FGF14, SYNE1, FOXD1, BMP6, SPRY1, TM4SF9, SNIP, STXBP6, TLL1, CSS3, OTP, TBR1, NRG1 and ITGA11 (). Real time RT-PCR to study gene expression was important in corroborating that hypermethylation is associated with the silencing of these genes. By using this validation method differences in expression were confirmed for SPRY1, NRG1, STXBP6, BMP6, CSS3, and SNIP among cells in different stages (). SPRY1, NRG1, STXBP6, BMP6, CSS3 and SNIP expressions were down regulated at different stages in the in vitro-in vivo
model when compared to the expression in MCF-10F cells (). The trMCF showed low expression of NRG1, STXBP6 and BMP6 compared to MCF-10F, and increased CSS3 (4.79 fold) and SNIP (1.89 fold) expressions. The invasive bsMCF cells showed low expression of SPRY1, NRG1, STXBP6, BMP6, CSS3 and SNIP. In caMCF, no expression of NRG1 and CSS3 was detected and these cells had low expression of BMP6, SPRY1 and SNIP however, they showed higher expressions of STXBP6 (). The expression studies did not correlate with the RLGS results for HOXA9, HOXB5, EPB49, FGF14, SYNE1, FOXD1, TLL1, OTP, TBR1, TM4SF9 and ITGA11; these genes showed the same level of expression at the different stages.
Restriction Landmark Genomic Scanning (RLGS)
Hypermethylated and hypomethylated genes in the in vitro- in vivo model of cell transformation induced by 17β-estradiol.
Expression studies in the in vitro-in vivo model of cell transformation
There are three active DNA methyltransferase (DNMTs) and DNA hypermethylation was correlated with their high expressions in tumors. Increased expression of the DNMT1 was found in colon tumors and leukemia (Smiraglia and Plass, 2002
) and its expression appears to increase progressively with advancing stages of both colon and lung cancers (Lee et al., 1996
). DNMT3A and DNMT3B expressions has been reported to be elevated in acute myeloid leukemia (Melki et al., 1998
) and in solid tumors (Belinsky et al., 1996
). The DNMTs expressions were studied in the in vitro-in vivo model of cell transformation using real time RT-PCR. The trMCF and caMCF4 cells showed higher expression of DNMT3B and DNMT3A respectively compared to MCF-10F (p<0.01) (). DNMT1 did not show significantly differences at the different stages of the in vitro-in vivo
DNMT1, DNMT3A and DNMT3B expressions in the in vitro- in vivo model of cell transformation