Introduction
Materials and methods
Cell culture and breast tissue specimens
Retroviral gene transfer
Immunoblot analysis
Cell cycle analysis
Chromosomal analysis
Centrosome analysis
Telomerase activity assay
Soft agar colony assay
In vivotumor studies
Methylation-specific PCR
Real-time quantitative PCR
Results
vHMEC are resistant to Ha-ras-induced proliferative arrest and display chromosomal abnormalities
Signals from the extracellular environment can cooperate with oncogenic ras to immortalize vHMEC and upregulate telomerase activity
Immortalized vHMEC expressing oncogenic ras are capable of anchorage-independent growth but are not tumorigenic in vivo
Progression to malignancy is associated with DNA methylation at several gene loci
Gene Name | Forward Primer (5'-3') | Reverse Primer (5'-3') | Product size (bp) | Primer location relative to TSS (bp) | Ref |
---|---|---|---|---|---|
p16INK4A-U | TTA TTA GAG GGT GGG GTG GAT TGT | CAA CCC CAA ACC ACA ACC ATA A | 151 | +132 to 283 | [22] |
p16INK4A-M | TTA TTA GAG GGT GGG GCG GAT CGC | GAC CCC GAA CCG CGA CCG TAA | 150 | +132 to 282 | |
HOXA9-U | TTG GGG TTA GAT AGG GAG TTG GGA | AAA AAT AAA AAC AAA AAA CAA ACA AA | 167 | -4453 to -4286 | [16] |
HOXA9-M | TCG GGG TTA GAT AGG GAG TCG GGA | AAA ATA AAA ACG AAA AAC AAA CGA A | 166 | -4453 to -4287 | |
CCND2-U | AGA GTA TGT GTT AGG GTT GAT T | ACA TCC TCA CCA ACC CTC CA | 108 | -1167 to -1059 | [12] |
CCND2-M | CGG CGA TTT TAT CGT AGT CG | CTC CAC GCT CGA TCC TTC G | 101 | -1138 to -1037 | |
RASSF1A-U | TTT GGT TGG AGT GTG TTA ATG TG | CAA ACC CCA CAA ACT AAA AAC AA | 108 | +201 to +309 | [23] |
RASSF1A-M | GTG TTA ACG CGT TGC GTA TC | AAC CCC GCG AAC TAA AAA CGA | 94 | +213 to +307 | |
SFRP1-U* | GTT TTG TAG TTT TTG GAG TTA GTG TTG TGT | CTC AAC CTA CAA TCA AAA ACA ACA CAA ACA | 135 | -2 to +133 | [24] |
SFRP1-M | TGT AGT TTT CGG AGT TAG TGT CGC GC | CCT ACG ATC GAA AAC GAC GCG AAC G | 126 | +2 to +128 | |
SFRP1-U** | TTT TAG TAA ATT GAA TTT GTT TGT GA | TAA AAT ACA CAA AAC TGG TAG AAC | 149 | -151 to -2 | [40] |
SFRP1-M | TTT AGT AAA TCG AAT TCG AAT TCG TTC GC | TAA AAT ACG CGA AAC TCC TAC G | 148 | -150 to -2 | |
p57-U | GTT GTT TGT GTT TGT GTA GTT TT | AAA AAT CCC ACA AAC AAC AAA ACA | 91 | +248 to +339 | |
p57-M | TTG TTC GCG TTT GCG TAG TTT C | AAA TCC CAC GAA CGA CAA AAC G | 88 | +249 to +337 | |
MGMT-U | TTT GTG TTT TGA TGT TTG TAG GTT TTT GT | AAC TCC ACA CTC TTC CAA AAA CAA AAC A | 93 | +30 to +123 | [25] |
MGMT-M | TTT CGA CGT TCG TAG GTT TTC GC | GCA CTC TTC CGA AAA CGA AAC G | 81 | +36 to +117 | |
THBS1-U | TTG AGT TTG TGT GGT GTA AGA GTA T | CCC CAC TAC CTA ACA CAC AAC T | 156 | -280 to -124 | [26] |
THBS1-M | GTT CGC GTG GCG TAA GAG TAC | CGC TAC CTA ACG CGC AAC T | 149 | -276 to -127 |
HMEC
|
vHMEC
|
vector
|
ras
|
ras0.5
|
184-A1
|
MCF-10A
|
MCF-7
|
MDA-231
| |
---|---|---|---|---|---|---|---|---|---|
p16
INK4A
| U |
M
|
M
|
M
|
M
|
M
|
M
|
M
|
M
|
HOXA9
| U |
M
|
M
|
M
|
M
|
M
|
M
|
M
|
M
|
CCND2
| U |
M
|
M
|
M
|
M
|
M
|
M
|
M
|
M
|
RASSF1A
| U | U | U | U | U/M |
M
|
M
|
M
|
M
|
SFRP1*
| U | U | U | U |
M
| U/M | U/M |
M
|
M
|
p57
| U | U | U | U |
M
| U |
M
|
M
| U |
MGMT
| U | U | U | U | U/M | U | U | U | U/M |
THBS1
| U | U | U | U | U | U | U | U | U |
Concomitant DNA methylation of RASSF1A and SFRP1occurs in malignant and premalignant breast lesions
Sample | Description | RASSF1A | SFRP1 |
---|---|---|---|
N1
| Normal reduction mammoplasty tissue | U | U |
N2
| Normal reduction mammoplasty tissue | U | U |
N3
| Normal reduction mammoplasty tissue |
M
|
M
|
N4
| Normal reduction mammoplasty tissue | U | U |
N5
| Normal reduction mammoplasty tissue | U | U |
N6
| Normal reduction mammoplasty tissue | U | U |
N7
| Normal reduction mammoplasty tissue | U | U |
Total Normal
Methylated
|
1/7
|
1/7
| |
H1
| Lobular hyperplasia (85% of mammary cells), blunt duct hyperplasia (15% of mammary cells) | U | U |
H2
| Normal (80%), intraductal hyperplasia (10%), cystic hyperplasia (10%) | U | U |
Total Hyperplasia
Methylated
|
0/2
|
0/2
| |
H3
| Focal atypical terminal duct hyperplasia (single focus); fibrocystic disease |
M
|
M
|
Total ADH
Methylated
|
1/1
|
1/1
| |
D1
| CCL, DCIS |
M
|
M
|
D2
| DCIS |
M
|
M
|
D3
| DCIS, cysts, normal breast tissue |
M
|
M
|
D4
| DCIS and normal tissue | U |
M
|
D5
| Extensive comedo DCIS, calcifications |
M
|
M
|
D6
| Extensive DCIS |
M
|
M
|
D7
| Extensive DCIS and normal tissue |
M
|
M
|
D8
| DCIS, calcifications | U |
M
|
D9
| Extensive DCIS |
M
|
M
|
D10
| DCIS with calcifications |
M
|
M
|
D11
| Extensive comedo DCIS |
M
|
M
|
Total DCIS
Methylated
|
9/11
|
11/11
| |
C1
| IDC, grade 2, node-, ER+ |
M
|
M
|
C2
| IDC, grade 3, node+, ER- |
M
|
M
|
C3
| IDC, grade 3, node+, ER- | U |
M
|
C4
| IDC, grade 3, node-, ER- |
M
|
M
|
C5
| IDC, grade 3, node+, ER+ |
M
|
M
|
C6
| IDC, grade 1, node+, ER+ |
M
|
M
|
C7
| IDC, grade 3, node+, ER- |
M
|
M
|
C8
| IDC, DCIS, CCL, normal breast |
M
|
M
|
C9
| IDC, DCIS |
M
|
M
|
C10
| IDC, DCIS, normal breast | U |
M
|
C11
| IDC and extensive DCIS |
M
|
M
|
C12
| IDC and extensive comedo DCIS with calcifications |
M
|
M
|
Total IDC
Methylated
|
10/12
|
12/12
|