sion between p. V600E and p. V600K. This would have had no impact on treatment i

Go down

sion between p. V600E and p. V600K. This would have had no impact on treatment i Empty sion between p. V600E and p. V600K. This would have had no impact on treatment i

Post  jy9202 on Mon Mar 10, 2014 10:11 am

er varies and both increased and decreased expression has been reported depending on the tissue of origin,In

breast cancer, strong BMP4 expression [You must be registered and logged in to see this link.] has

been found in both cell lines and tissues and immunohistochemical data indicate that BMP4 protein is expressed

in one fourth to half of primary tumors,Functional studies in multiple malignancies suggest that BMP4 typically

causes reduced growth and increased migration of cancer cells,We have previ ously shown, using a large set of

breast cancer cell lines, that BMP4 treatment systematically inhibits proliferation in all cell lines and

simultaneously increases migration of MDA MB 231, MDA MB 361 and HCC1954 cells, but reduces migrativeness of T

47D cells,Similarly, Guo and colleagues demonstrated increased migration and decreased proliferation upon BMP4

overexpression in MDA MB 231 and MCF 7 breast cancer cells.

@ These data were corroborated by an in vivo

study where inhib ition of BMP4 signaling decreased metastasis of MDA MB 231 breast cancer cells,Yet there is

one study where BMP4 reduced migration of MDA MB 231 cells,Nevertheless, the majority of the data implies that

BMP4 has a dualist effect on breast Lenalidomide

Revlimid
cancer cells, with inhibition of cell proliferation and induction of a migratory phenotype. The

aforementioned in vitro functional studies were done using cells growing as two dimensional mono

layer.

@ However, there is an increasing interest in culturing cells in a more biologically relevant

three dimensional environment,This has been generally achieved by growing cells in synthetic scaffolds or gels

of biological or synthetic origin,Matrigel, basement membrane extract from mouse sarcoma, is the most

[You must be registered and logged in to see this link.] commonly used biological scaffold

and consists mainly of laminin, collagen IV and various growth factors,Other biological mate rials that are

often used include collagen, alginate and hyaluronic acid,Synthetic gels have been developed as alternatives to

the biological gels due to the difficulties in defining the exact composition of the biological mate rials and

the fact that they may suffer from batch to batch variability,Synthetic gels, mainly different polymers, such

as polyethylene glycol and polyvinyl alcohol, have a constant composition and are easy to manipulate.

@

However, they may not adequately represent the com plicated extracellular matrix that surrounds cells in

tissues,Various cell types, including epithelial, neural and endo thelial cells, have been successfully grown

in 3D and are capable of forming structures that resemble the normal tissue organization,For example, normal

immortal ized mammary epithelial cells, such as the MCF 10A cells, form polarized acini structures in Matrigel,

reminiscent of the normal breast architecture,whereas breast cancer cells generate more variable

structures,Similarly, biologically appropriate cellular organization has been observed e. g.

@ for

epithelial and neural cells in different synthetic gels,More importantly, the shift from 2D to 3D culture also

results in changes in gene expres sion in multiple tissue types,For example, breast epithelial cells begin to

produce milk proteins when grown in Matrigel,Previous data from us and others showed that BMP4 is able to

reduce the growth of breast cancer cells whilst inducing cell migration and invasion,Here we utilized two

different 3D culture systems to evaluate whether these phenotypes persist under more physio logical culture

conditions and further explored the mechanisms of BMP4 induced changes in cell prolifera tion and mobility.@

Methods Cell lines The MCF 10A, MDA MB 231, MDA MB 361, BT 474 and T 47D cell lines were purchased from ATCC

and cultured according to ATCC instructions, except for MCF 10A, which was maintained as previously

described,In 3D experiments, MDA MB 231 and MDA MB 361 cells were cultured in DMEM,For MCF 10A cells a reduced

concentration of EGF was used in Matrigel,BMP4 and inhibitor treatments rhBMP4,BMP antagonist Gremlin,MMP

inhibitor Batimastat or a combination of these was added to the medium at the start of the experiments and rep

lenished every two to three days as the medium was exchanged.

Vehicle treated cells received BMP4

dilution buffer,Gremlin dilution buf fer,Batimastat dilution buffer,or a combination of these. All experiments

were done in two to six replicates and were repeated at least twice. Cell proliferation assay Medium with 10%

alamarBlue was added to the cells and incubated for 1 hour or 4 hours,Medium was collected and fluor escence

measured using Tecan infinite F200 Pro plate reader,Addition ally, the number of cells in 2D culture was

counted using the Z1 Coulter Counter at indicated time points.@ The experiments were done in four to six

replicates and repeated at least twice. Cell cycle MCF 10A cells were cultured on 24 well plates and analyzed 3

and 5 days after first addition of BMP4. The cells were stained with PI as described,The cell cycle

distribution was determined using the Accuri C6 flow cytometer and ModFit L

jy9202

Posts : 509
Join date : 2013-12-18

View user profile

Back to top Go down

Back to top

- Similar topics

 
Permissions in this forum:
You cannot reply to topics in this forum