Biologia Cel·lular 2 2022-2023 Name: Anna Aguilera Romero Problem 2

A patient was diagnosed with prostate cancer and a biopsy of the tumor was taken. From the
epithelial cells of the biopsy, a cell line was established (Prost-A) that, upon genetic and
transcriptomic analysis, the investigators concluded that it was very similar to the primary
tumor. After two years of treatment the patient, unfortunately, relapsed. Then, a second
biopsy of the tumor was taken and a second cell line (Prost-B) was established. Genomic
sequencing and transcriptomic analysis showed many changes with respect to the original
tumor and cell line. Meanwhile, in the laboratory other tests were done to find out relevant
differences between the two cell lines.

Figure 1. The cell lines Prost-A (A) and Prost-B (B) were treated with 10nM androgen
(testosterone) for the indicated times and the androgen receptor (AR) was detected by
immunofluorescence. Cells were counterstained with DAPI to visualize the nuclei.
Biologia Cel·lular 2 2022-2023 Name: Anna Aguilera Romero Problem 2

Figure 2. Nuclear extracts (NEXT) were prepared from the cell lines Prost-A and Prost-B
without any treatment (-Tes) of after 60 minutes of treatment with 10nM testosterone (+Tes)
and binding of the AR was tested by Electrophoretic mobility shift assay (EMSA) using a labeled
probe containing two inverted HRE repeats. When indicated, a monoclonal antibody against
AR (AR mAb) was included in the binding reaction.
Biologia Cel·lular 2 2022-2023 Name: Anna Aguilera Romero Problem 2

Figure 3. A low-density colony forming assay was performed with cells from Prost-A (cell line
A) and Prost-B (cell line B) in the presence (+Tes) or the absence (-Tes) of 10nM testosterone.
Cells were plated at low density and after 7 days, the tissue plates were stained with crystal
violet.

Questions (very brief answers).

1. Explain the different behavior of the AR in the Prost-A and Prost-B cell lines upon
testosterone treatment as deduced from Figure 1.

As we can deduce from Figure 1, AR has nuclear function. In case of cell Prost-A line, when it
binds its ligand (androgen), it translocates to the nuclei, that is why the fluorescence tends to
concentrate in the nuclei as time goes by. Otherwise, in case of Prost-B, the AR is in the nuclei
since the beginning of the experiment and doesn’t change in time.

2. From the EMSA assay from Figure 2:

What is the purpose of including a monoclonal antibody against AR in the binding reaction?
Including a monoclonal antibody against AR captures the receptor and shows us that we are
seeing the androgen receptor, not other random protein.
What would be the results if a probe containing two direct HRE repeats would be used?
There would be no results because the AR binds to inverse HRE repeats instead of binding two
direct HRE repeats.
What would be the results if cytosolic extracts instead of nuclear extracts obtained in the
same conditions would be used?
In the case of Prost-A, we would see the bands in the not treated lane due to the AR doesn’t
translocate if there are no androgens. Otherwise, in the case of Prost-B, we wouldn’t see
Biologia Cel·lular 2 2022-2023 Name: Anna Aguilera Romero Problem 2

anything (even if they are treated or not) because the AR is inside the nuclei from the
beginning, there is not AR in the cytosol and it doesn’t have to translocate.

3. Explain the results of Figure 3.

In the case of Prost-A, cells need androgen to translocate the AR to the nuclei, that is why in
the presence of testosterone there is growth, but the in absence of it, there is not growth.

Otherwise, in the case of Prost-B, AR is already in the nuclei, and it has constitutive activity,
that is why the cells grow in presence and in the absence of testosterone.

4. Which would be the consequences of the conclusions of these experiments for the
treatment of the prostate cancer patient?

This prostate cancer in Prost-B has a constitutive activity of AR, the treatment should be
focused on inhibit this receptor maybe with an allosteric inhibitor or with an HRE blocker that
cuts the activation pathway.