Here is a reference:

MCF-7 breast cancer cell line obtained by long term treatment of the wild type MCF-7 cell line with 4-hydroxytamoxifen (4-OH Tam). METHODS: We cultured MCF-7 cells with 4-OH Tam over a period of 12 months to obtain the resistant cell line.

Title: Proteomic analysis of acquired tamoxifen resistance in MCF-7 cells reveals expression signatures associated with enhanced migration.

Author: Zhou C; Zhong Q; Rhodes LV; Townley I; Bratton MR; Zhang Q; Martin EC; Elliott S; Collins-Burow BM; Burow ME; Wang G

Journal: Breast Cancer Res; 2012 Mar; 14(2):R45. PubMed ID: 22417809

Good luck!

What passage number are you using and are you adding TAM to the media to maintain resistance? after many passages, resistant cell lines can transform. also, to maintain resistance you probably need to maintain a low dose exposure of TAM in the cultivating media. but as Rolando noted above, it does take a year or more to establish resistance so you may need to verify the initial resistance of your cell line with a dose response study.

I got the cells from another laboratory and they told me that they are maintaining that cells for a long time. and i'm maintaining this resistant cells in DMEM media with 3 µM Tamoxifen. But when i add 15 µM tam to the cells, it seems the concentration is almost the IC50 dosage. For MTT assay and crystal violet assay, I got the value below 50. And at the same time, i'm maintaining normal MCF-7 cells also, and it having an IC50 dosage of tamoxifen of 25 µM. I'm in confusion for those cells that, which one is tamoxifen resistant.

And thank you for both Ronaldo and Lara for giving me advice. And i need more help about these.

As a starting point, i would suggest doing parallel dose response studies with both cell lines, with a high number of replicates, positive and negative controls. Also account for the slower double time generally exhibited by resistant cells. An alternative starting point, of course, would be to obtain a new vial of cells if accessible!

The growth of tamoxifen resistant MCF-7 cell line is much slower when comparing to the normal MCF-7 cell lines. Which means it should be a resistant cell. But the presence of tamoxifen in the media will also affect the growth and doubling time. I'm also planning to but new vials of resistant cells.

The best way to deal with this is to perform a kill curve against parental MCF7 to gauge whether the specific mcf line you are working with is indeed resistant. Most of the time, resistant cell lines are not clonal and consist of a population of cells with varying degrees of resistance so if the problem persists I would take the time to single cell clone (while under the influence of tamoxifen of course). Also, be careful when you dissolve your 4oht; since it is soluble in ethanol you may want to make your stock solution as concentrated as possible, I've seen people "fix" their cells by having too much ethanol in their media. Also, have you been maintaining the cell line with 4oht? It takes as little as two weeks without tamoxifen for a cell line to lose resistance to the drug. In my experience if you are comparing against a parental mcf7 that is over 150 passages you might as well get a new vial of parentals; their growth rate nearly triples obscuring your results.

The levels of 4OHT you are using are extremely high! You might be dealing with non ER mediated effects such as chemical toxicities. Tam is still a chemical and may influence irrelevant chemical reactions in the cell. Non-resistant MCF-7 cells arrest with 100nM 4OHT so perhaps you should consider lowering your dose and as mentioned comparing it against the parental cell line.

When adapting MCF-7 cells or any other ER+ cell line to conditions of low estrogen or anti-estrogens (SERMS or SERDS), it is important to use IMEM without phenol red and supplemented with 10% Charcoal-stripped calf serum (CCS). 10% fetal bovine serum contains enough estrogen to saturate ER and would therefore require a higher concentration of tamoxifen to displace estrogen. Further, phenol red is estrogenic. I have generated a tamoxifen and fulvestrant resistant lines of MCF-7 as follows. I seed wild-type MCF-7 in a T-75 in 10% FBS. 24 hours after seeding, I start to deplete the cells of exogenous estrogens (found in serum as well as the phenol red) by washing the cells with 5 cycles of the following: aspirate the medium and rinse with IMEM (no phenol red, no serum) two times and then return the cells to IMEM (no phenol red) supplemented with 10% charcoal stripped serum. Each cycle is repeated once per hour for five hours for three days. At this time, the cells will be free of exogenous estrogens and the residual effects of estrogen will have ceased. Now, you can culture your cells in IMEM (no phenol red) supplemented with 10% CCS and tamoxifen. I use the IC90 of tamoxifen; that is the dose of tamoxifen required to displace estrogen such that 90% of the cells are dead. Over a long haul of 6-12 months, the cells will become resistant to the tamoxifen and start to grow again. Eventually, tamoxifen-resistant cells will grow as quickly as wild-type MCF-7. Once a month, you can do a kill curve comparison between the adapted cell lines and the parental cell line in response to tamoxifen AND, you can do a growth response to estrogen. Several mechanisms have been proposed for tamoxifen resistance ranging from increased expression of growth factor receptors, auto-phosphorylation of estrogen receptor to estrogen receptor recognizing tamoxifen as a growth stimulus and recruiting similar co-activators as estradiol. Hope this helps...

jose

Addendum: In our lab, we are able to induce maximal growth in MCF-7 with 100pM estradiol (E2). Likewise, we are able to fully antagonize E2-induced growth with 100nM tamoxifen. Alternatively, we can also antagonize E2-induced growth with 10nM endoxifen. So, my tamoxifen-resistant MCF-7 cells were established with 100nM tamoxifen. My stock of tamoxifen is 10mM in 100% ethanol. Do the math and my % ethanol in the final tamoxifen medium is way under 0.1%, read: negligible.

You may wish to follow up the work of my colleagues Dr Julia and Prof Bob Nicholson in Cardiff using the following weblink of their latest work on long term cultures of tamoxifen resistant MCF-7 cell lines

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3393679/

ask to Etienne Leygue, one of my followers and followings. He is a specialists of MCF7...

Actually, I'm using the tamoxifen dissolved in DMSO. And in some research papers, they used tamoxifen concentration of 8 µM for 72 hours. So I tried that for 24 hrs and got the concentration as 25 µM by MTT assay and crystal violet assay. And for normal MCF-7 cell line, I got this concentration. But while checking in tamoxifen resistant MCF-7 cell line, I got the IC50 dosage below 15 µM.

To work with MCF-7 and estradiol or tamoxifen, MTT /WST-1 are not appropriated, use cell counting approach only. It is much more laborious but the only way to prevent the issues you have.

So now you know that resistance Tam could mean just one Log or less shift in sensitivity to Tam. This is observable for acquired as well as intrinsic resistance to Tam. Mechanisms of growth control are not just a matter of estrogen recemptors but can involved other pathways such as cholesterol metabolism, that are targetted by Tam and are involved in its mechanism of growth control and cytotoxicity

I got the doubling time for Tam resistance ( MCF/7) less/lower than the parental , is that normal ? as some articles showed a low growth rate of the Tam resistance

Yes initially it will be less than wild type parental,once you continue them grown in tamoxifen,and the time they become resistant ,the doubling time will be same as wild type.

Tamoxifen metabolism in vivo requires the action of CYP450 (2D6 in particular) This is inhibited by many commonly and concomitantly prescribed drugs. In poor or non metabolisers it may not be a resistant cells that are causing the chemo to fail but an innate or induced inability to metabolise the drug. There is a world of difference between in vitro and in vivo studies to which insufficient attention is being paid.

If a resistant clone of tamoxifen is generated, how frequently kill curve should be checked? every revival or once in a month?

If a resistant clone is generated with 4-oh tamoxifen, what happens if we treat it with tamoxifen. does it behave the same or different? 

Does any one has the problem of easy detachment of MCF Tamoxifen resistance cell from culture dish? I am new to this cell line and growth  is also too low. I am not sure whether its common?