Background
Methods
Materials
Cell culture
Source and Maintenance
Selection Process
Fixation and staining of selected MCF10AT1 cells
Proliferation test assay
mRNA analysis of gene expression
c-Neu : 5'-AGC TGG TGA CAC AGC TTA-3' FP
[8] 5'-TGG TTG GGA CTC TTG AC-3' RP
ERα 5' CAA GCC CGC TCA TGA TCA A-3' FP
(228 bp) 5'-CTG ATC ATG GAG GGT CAA ATC CAC-3' RP
Cyclin D1 5' GGA TGC TGG AGG TCT GCG A-3' FP
(146 bp) 5'-AGA GGC CAC GAA CAT GCA AG-3' RP
p21 5'-ATT AGC AGC GGA ACA AAG AGT CAG ACA T-3' FP
(318 bp) 5'-CTG TGA AAG ACA CAG AAC AGT ACA GGG T-3' RP
p27 5'-AAC GTG CGA GTG TCT AAC GG-3' FP
(164 bp) 5'-CTT CCA TGT CTC TGC AGT GC-3' RP
VEGF 5'-AAG GAG GAG GGC AGA ATC AT-3' FP
(226 bp) 5'-ATC TGC ATG GTG ATG TTG GA-3' RP
IGF-IR 5'-ACA GAG AAC CCC AAG ACT GAG G-3' FP
(285 bp) 5'-TGA TGT TGT AGG TGT CTG CGG C-3' RP
c-met 5'-CAG GCA GTG CAG CAT GTA GT-3' FP
(201 bp) 5'-GAT GAT TCC CTC GGT CAG AA-3' RP
CK19 5'-CTA CAG CCA CTA CTA CAC GAC-3' FP
(148 bp) 5'-CAG AGC CTG TTC CGT CTC AAA-3' RP
MMP-12 5'-ACA CAT TTC GCC TCT CTG CT-3' FP
(192 bp) 5'-CCT TCA GCC AGA AGA ACC TG-3' RP
MMP-13 5'-AAC ATC CAA AAA CGC CAG AC-3' FP
(166 bp) 5'-GGA AGT TCT GGC CAA AAT GA-3' RP
NFκB 5'-CAC TTA TGG ACA ACT ATG AGG TCT CTG G-3' FP
(406 bp) 5'-CTG TCT TGT GGA CAA CGC AGT GGA ATT TTA GG-3' RP
β-Actin 5'-GGA CTT CGA GCA AGA GAT GG-3' FP
(234 bp) 5'-AGCACT GTG TTG GCT TAC AG-3' RP
MMTV mice treatments
Statistical Analysis
For proliferation and mRNA expression experiments
For MMTV mouse tumor studies
Results
Observations on phenotypic changes in MCF10AT1 cells exposed to chemicals for 20 passages
Cellular morphology in a confluent system
Changes in baseline cellular proliferation
Changes in mRNA expression (Table 1)
MCF10AT1 sublines selected for 20 passages in DMEM/F12 media containing: | ||||
---|---|---|---|---|
Control | E2 (1 nM) | Tam (1 nM) | β-HCH (1 uM) | |
Proto-oncogenes
| ||||
cNeu | 1.00 | 1.64* | 1.79* | 2.73** |
Cyclin D1 | 1.00 | 1.84* | 1.88* | 2.50** |
p21 | 1.00 | 2.05* | 1.07 | 1.19 |
p27 | 1.00 | 2.02* | 1.73* | 2.80* |
Cell Status
| ||||
VEGF | 1.00 | 3.12* | 0.72 | 1.34 |
IGF-IR | 1.00 | 1.99* | 2.79* | 3.77** |
c-met | 1.00 | 2.06** | 0.66* | 1.26 |
CK19 | 1.00 | 0.78* | 0.72* | 1.67* |
Invasive
| ||||
MMP-12 | 1.00 | 0.85 | 0.41* | 3.50* |
MMP-13 | 1.00 | 0.86 | 18.51** | 14.12** |
Inflammation
| ||||
NFkB | 1.00 | 3.20** | 0.24* | 2.59** |