Functional roles of chromosomes 11 and 17 in the transformation of human breast epithelial cells in vitro.
Genomic alterations in primary breast cancer play a role in the initiation and progression of the disease. We have analyzed the molecular events involved in the initiation and progression of the neoplastic process in an in vitro experimental system. Immortalization of human breast epithelial cells (HBEC) is associated with 3:9 translocation, p53 mutation and microsatellite instability (MSI) of chromosomes 11p13, and 17p. BP1-E cells, derived from the immortalized MCF-10F cells transformed by the carcinogen benzo(a)pyrene (BP), express in vitro growth advantage, anchorage independence, enhanced chemoinvasiveness, loss of ductulogenic capabilities and tumorigenesis in a heterologous host. This neoplastic progression is also associated with mutations and/or amplification of c-H-ras, int-2, c-neu, c-myc and MDM2, MSI at 11q25 and 13q12-q13 and loss of heterozygosity at 17p. In order to test whether chromosomes 11 or 17 play a functional role in the phenotypic expression of transformation of BP1E cells, we utilized microcell-mediated chromosome transfer (MMCT) technique for inserting the corresponding normal chromosomes to these transformed cells. BP1E cells were transfected with PsV2neo plasmid and fused with microcells obtained from the mouse cell line A9, containing a normal chromosome 11 or 17 (A9-11neo and A9-17neo cells, selected in G418 and cloned. Sixteen primary microcell hybrids from each chromosome transfer, designated BP1E-11neo and BP1E-17neo survived selection in G-418 containing medium. A single clone from each group, BP1E-11neo #145 and BP1E-17neo D100, survived subcloning and were utilized for a detailed panel of analyses. The presence of a donor chromosome was confirmed by dual color fluorescence in situ hybridization (FISH), southern blot analysis of the marker vector pSV2neo, and microsatellite polymorphism analysis. The transfer of the normal chromosomes 11 and 17 resulted in a 50% and 90% inhibition of cell growth respectively, and reduced both colony efficiency and colony size. Telomerase activity was significantly reduced only by chromosome 17 insertion, providing a possible explanation for the more significant senescence observed in BP1E-17neo D100 cells. Microsatellite polymorphism analysis revealed that three loci, 11q13-23, 11q23.1, and 11q23.3 (markers D11S911, DRD2, and D11S29) were retained in BP1E-11neo #145 cells, and two, 17q24.2-25.2, 17q25.2 (markers D17S515 and D17S785 were retained in BP1E-17neo D100 cells. We conclude that the specific regions of normal chromosomes 11 and 17 transferred play a functional role in the expression of immortal and transformed phenotypes of HBEC in vitro.
Yang
X
X
Breast Cancer Research Laboratory, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Tahin
Q
Q
Hu
Y F
YF
Russo
I H
IH
Balsara
B R
BR
Mihaila
D
D
Slater
C
C
Barrett
J C
JC
Russo
J
J
D015139:Blotting, Southern; D001940:Breast / Q000166:cytology*; D002455:Cell Division / Q000235:genetics; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002478:Cells, Cultured; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics*; D002886:Chromosomes, Human, Pair 17 / Q000235:genetics*; D002999:Clone Cells / Q000166:cytology / Q000201:enzymology; D004847:Epithelial Cells / Q000166:cytology* / Q000201:enzymology; D006801:Humans; D006822:Hybrid Cells / Q000166:cytology; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D018895:Microsatellite Repeats / Q000235:genetics; D010641:Phenotype; D016133:Polymerase Chain Reaction; D019098:Telomerase / Q000378:metabolism; D014162:Transfection; D014410:Tumor Stem Cell Assay
D019098:Telomerase