CWR22Pc cell line is derived from the androgen-dependent re-transplantable primary CWR22 tumors
CWR22 prostate tumor system was originally established from a Gleason score 9 human prostate cancer obtained from prostatectomy. The prostate cancer tissue pieces were grown in male nude mice as subcutaneous tumors and the tumor system is maintained by serial re-grafting. The tumors were named primary CWR22 tumors, and they are dependent on androgens for growth. In response to long-term androgen deprivation, the tumors regress but recur back within 7-9 months as androgen-independent tumors. The androgen-independent secondary tumors were named recurrent CWR22 tumors and a cell line established from the recurrent CWR22 tumors was named CWR22Rv1.
In this work, we established a new prostate cancer cell line from the primary CWR22 tumors. In order to confirm the genetic lineage identity of CWR22Pc cell line as a derivative of the primary CWR22 tumors, we compared the DNA fingerprinting pattern of two passages of CWR22Pc line with that of the CWR22 primary tumors at nine different genetic markers (8 STR markers and the Amelogenin locus). CWR22Pc cell line showed an identical DNA fingerprinting pattern to the primary CWR22 tumors (number of alleles and allele size) at all 8 STR markers. The Y-specific Amelogenin locus showed two alleles (size 213 and 219 bp) in both CWR22Pc cells and in the primary CWR22 tumors indicating the presence of an X and a Y chromosome. show a comparison between the results of DNA fingerprinting analysis of the primary CWR22 tumor and the two passages of CWR22Pc cells (#6 and #21) at the 9 loci studied. Moreover, we compared the chromosomal alterations in the CWR22Pc line with the ones in the primary CWR22 tumor. Specifically, we evaluated DNA obtained from two passages of CWR22Pc cells and DNA from the primary CWR22 tumor using comparative genomic hybridization (CGH). CGH analysis is an ideal method to detect overall chromosomal gains and losses in the genome. The CGH analysis showed that the pattern of chromosomal alteration of the CWR22Pc cell line was very similar to that of the primary CWR22 tumor. Chromosomal gains included gains at 1q, 7, 8p and 12, and losses of chromosomes 2 and X (). The findings of the DNA fingerprinting analysis and CGH confirmed that the CWR22Pc cell line was indeed derived from the primary CWR22 tumors. In order to evaluate the ploidy status of the CWR22Pc cells, we performed a G-banding analysis of over 300 metaphase spreads and a ploidy analysis by flow cytometry. Metaphase analysis showed that approximately 80% of the cells were near tetraploid (chromosomal count approximately 100), and 20% of the cells showed a chromosomal count of approximately 50 chromosomes. These findings were consistent with the cell cycle and ploidy analysis which showed a mixed population of cells with DNA content of 1.11 and 2.23 ().
The genetic lineage identity of CWR22Pc cells is similar to that of CWR22 primary prostate tumors
In summary, our results of the G-banding and the ploidy analysis of the CWR22Pc cells are in line with the mosaic karyotype reported previously for the primary CWR22P tumors (6
), the recurrent CWR22R xenografts (14
) and the CWR22Rv1 cell line established from the recurrent CWR22R tumors (8
). In detail, primary CWR22 xenograft tumors were reported to have a mixture of two karyotypes where part of the cells displayed an unidentified marker chromosome (6
). In addition, the relapsed strains of CWR22R tumors after androgen deprivation showed each a different karyotype (14
), and the CWR22Rv1 cell line consists of a mixed population of hyperdiploid (90%) and near-tetraploid (10%) cells (15
). The results of our study reported here indicated a mosaic karyotype of CWR22Pc cells. It will be both important and interesting for the future studies to determine whether the ploidy status of CWR22Pc cells will change during a long term androgen deprivation in vitro
and when CWR22Pc cells are grown as xenograft tumors in nude mice in vivo
Growth and PSA protein expression of CWR22Pc cells is regulated by androgens
Given that the CWR22Pc cell line was established from primary CWR22 tumors, which are regulated by androgens, we first aimed to determine the effects of androgens on the growth of CWR22Pc cells in culture. CWR22Pc cells, LNCaP cells and androgen-independent CWR22Rv1 cells were cultured in the presence or absence of 0.8 nM dihydrotestosterone (DHT) for nine days, and the number of attached viable cells was determined every third day. On day 6 of the experiment, the number of CWR22Pc cells was increased by 3-fold, whereas the number of LNCaP cells was increased only by 40% (, panel i). Moreover, on day 9 of the experiment, the number of CWR22Pc cells was increased by 6-fold by androgens while the number of LNCaP cells was increased only by 60% (, panel i). At the same time, the growth rate of CWR22Rv1 cells was not significantly affected by DHT during a 9-day period. Deprivation of CWR22Pc cells from androgens induced apoptotic death of the cells as demonstrated by cell morphology on the day 9 of the experiment (, panel ii). Specifically, androgen deprivation induced extensive detachment of the cells, cell fragmentation, shrinkage and blebbing, all of which are morphological changes consistent with apoptotic cell death (, panel ii). Moreover, DNA fragmentation was increased by 5-fold on the day 3 of the experiment in the cells cultured in the absence of DHT (, panel iii).
The growth of CWR22Pc cells is increased by androgens
To further examine androgen regulation of CWR22Pc cells in culture, we compared the effect of DHT on PSA protein expression in CWR22Pc vs. LNCaP or CWR22Rv1 cells. The promoter region of PSA is known to contain an androgen response element. The cells were cultured in the presence or absence of 0.8 nM DHT, and the levels of PSA protein in whole cell lysates were determined by western blotting. The PSA protein levels were increased by 3-fold in CWR22Pc cells when the cells were cultured in the presence of DHT (). DHT increased PSA protein expression in LNCaP cells by approximately 2-fold. The levels of PSA protein in CWR22Rv1 cells were undetectable when compared to both CWR22Pc and LNCaP cells ().
CWR22Pc cells are highly tumorigenic in nude mice and the tumor growth is regulated by androgens
Once we had established that CWR22Pc cells were genetically similar to the primary CWR22 tumors and the growth of CWR22Pc cells in culture is regulated by androgens, we next focused on evaluating whether CWR22Pc cells are tumorigenic in athymic nude mice and whether in vivo growth of CWR22Pc cells is regulated by androgens. To investigate whether CWR22Pc cells would grow as xenograft tumors in nude mice, we injected CWR22Pc cells (20 x 106 cells per site) subcutaneously to the flanks of athymic nude mice (n=20) (two tumors per mouse). The nude mice were castrated and half of the mice (n=10) were implanted with sustained-release 5α-DHT-pellets to normalize the circulating androgen levels. In the mice supplied with DHT-pellets, tumors started to form on day 7 with a 100% incidence (, panel i). Importantly, the tumors in mice supplied with the DHT pellets grew rapidly, while both the tumor incidence and the growth rate were low in mice without DHT pellets (, panel i). These results suggested that CWR22Pc human prostate cancer cells are highly tumorigenic in athymic nude mice and the tumor growth is regulated by androgens.
Since CWR22Pc cells in culture produced high levels of PSA, and the PSA protein expression was regulated by androgens, our next aim was to investigate whether serum PSA levels would correspond with the volumes of the CWR22Pc xenograft tumors in mice. Serum PSA levels were determined from the blood samples collected on day 27 of the experiment (, panel ii) from the athymic nude mice carrying CWR22Pc tumors presented in , panel i. The results of the serum PSA assay showed that PSA protein levels in serum of the mice closely correlated with the volumes of the CWR22Pc subcutaneous xenograft tumors (, panel ii). Specifically, serum PSA levels were almost undetectable in mice without DHT pellets carrying small tumors at low incidence. In contrast, serum PSA levels closely followed tumor volumes in DHT-supplied mice which developed large CWR22Pc xenograft tumors with a high incidence (, panel ii; the tumor volume is the mean of the two tumors in each mouse). Collectively, the results presented here indicate that the cells in subcutaneous CWR22Pc xenograft tumors secrete high levels of PSA to the circulation of the mice. Moreover, the serum PSA levels in these mice correlate with the volumes of the xenograft tumors.
CWR22Pc tumors recur after androgen withdrawal
In the next set of experiments, we first grew CWR22Pc cells as subcutaneous athymic nude mice supplied with DHT-pellets two consecutive experiments (n=34 mice, 1 tumor per mouse and n=30, 2 tumors per mouse). When the tumors reached 10 mm in diameter in size, the DHT pellets were removed and the tumor growth was followed by consecutive tumor volume measurements. The removal of the DHT pellets resulted in both experiments in a regression which reached the maximum in 15 days (). Importantly, the tumors re-grew back in the androgen-deprived nude mice with the following 20-30 days ().
The human prostate cancer cell line CWR22Pc established from the primary CWR22 tumors provides several valuable advantages as a model system for androgen-regulated growth and development of androgen-independence of prostate cancer cells. First, androgen-promoted tumor growth of CWR22Pc cells inoculated as subcutaneous tumors in nude mice occurs fast (in 3 weeks) with 100% tumor incidence. Second, development of hormone-refractory tumors is highly reproducible and the tumor re-growth takes place rapidly. Third, the CWR22Pc tumor system allows easy genetic in vitro genetic manipulation of the cells that form the tumors.
CWR22PC cells express androgen receptors
To analyze the AR protein expression level in CWR22Pc cells, we immunoblotted CWR22Pc, LNCaP and CWR22Rv1 cell lysates for androgen receptor using an anti-human AR mAb (). The filters were stripped and re-blotted with anti-actin pAb to demonstrate the loading of total proteins. The results show that AR protein is expressed at a high level in all three cell lines. Moreover, the results demonstrate that the size of AR in CWR22Rv1 cells is slightly larger compared to the AR in LNCaP and CWR22Pc cells. This was the expected result since the relapsed CWR22 tumors (CWR22R) and the CWR22Rv1 cell lines established from the relapsed tumors are known to express an AR gene that contains an in-frame tandem duplication of the exon 3. This region encodes the second zinc finger of the AR DNA-binding domain and the AR protein product has an approximately 5-kDa increase in protein size relative to the LNCaP AR (16
). Moreover, the AR gene in the relapsed CWR22 tumors and in CWR22Rv1 cells has the H874Y mutation in the ligand binding domain of AR, which enables the receptor to bind adrenal androgen dehydroepiandrosterone in addition to estradiol, progesterone and hydroxyflutamide (17
) (). The AR gene also in the primary CWR22 tumors contains the H874Y (histidine to tyrosine) mutation. However, the AR gene in the primary CWR22 tumors does not have the tandem duplication in the exon 3 present in CWR22Rv1 ().
Androgen receptor expression in CWR22Pc cells
As the next step, we wanted to characterize the AR transcript in CWR22Pc cells by RT-PCR mapping and by sequencing to determine whether the AR gene in CWR22Pc cells contains the H874Y mutation found in the AR in the primary CWR22 tumors. Moreover, we wanted to determine whether the AR gene in CWR22Pc cells contains any additional mutations. To that end, we performed RT-PCR mapping using primer pairs designated to amplify overlapping AR mRNA segments of approximately 600-900 bp and spanning the entire length of the AR coding sequence. The exons and the 5′- and 3′-untranslated sequences amplified by each of the primer sets are described in . RT-PCR reactions performed using RNA isolated from primary CWR22Pc cells, primary CWR22 xenograft tumors (CWR22P), LNCaP cells and CWR22Rv1 cell line yielded expected sizes for the amplification products of the segments I, II, and IV of the AR gene. The RT-PCR product for the segment III, which spans the exon 3 of the AR gene, yielded the expected amplicon size of 673 bp in CWR22Pc cells, primary CWR22 xenograft tumors and in LNCaP cells, while the CWR22Rv1 RT-PCR reaction yielded an approximately 100-bp larger RT-PCR product due to the exon 3 tandem duplication as reported previously (16
) (). We then purified the segments I-IV amplification products obtained from CWR22Pc cells and analyzed them automated DNA sequencing followed by ClustalW-driven pairwise alignment with the reference AR cDNA sequence in GenBank (National Center for Biotechnology Information). The sequence comparison demonstrated the H874Y mutation in the AR gene in CWR22Pc cells, while no additional mutations in the AR gene were found in CWR22Pc cells. In summary, these results demonstrated that the sequence of the AR gene in the CWR22Pc cells has the H874Y mutation but not the exon 3 duplication. Moreover, the data indicate that the AR gene is identical to that in the primary CWR22 xenograft tumors, which indirectly supports the notion that CWR22Pc cell line is derived from the primary CWR22 tumors, and is genetically different from the androgen-independent CWR22Rv1 cell line.
Currently, only few androgen-dependent human prostate cancer cell lines exist such as LNCaP (3
), LAPC-4 (18
) and MDA PCa 2b (13
). Androgen-independent cell lines PC-3 (20
) and DU145 (21
) were derived from metastatic lesions to the bone and brain, respectively. Moreover, CWR22Rv1 human prostate cancer cell line (8
) is independent of androgens for growth and was established from a xenograft tumor derived from an untreated primary prostate cancer (5
). It is important to note that AR expression persists in clinical prostate cancer despite progression to hormone-refractory state (22
). AR in prostate cancer typically undergoes genetic alterations including AR gene amplification during hormone therapy. Moreover, 10-30% of prostate cancers acquire a point mutation in the AR gene (23
). Both types of known AR gene alterations lead to increased sensitivity of the receptor to low levels of circulating androgens and also to the receptor’s ability to recognize a broadened spectrum of ligands as potent agonists of AR action. DU145 and PC-3 cells are both AR-negative prostate cancer cell lines, whereas LNCaP, MDA pCa 2b, LAPC-4 and CWR22Rv1 cells express AR protein. In LNCaP cells, the androgen receptor has a point mutation T877A which makes the AR more sensitive to flutamide, estradiol and progesterone (24
). LAPC-4 cells express wild-type AR, while AR in MDA PCa 2b cells contains two mutations, T877A and L701H, which reduces the AR affinity to androgens but enhances binding of adrenal corticosteroids to the AR (25
). CWR22Rv1 cells are AR-positive, but the AR contains a mutation H874Y in addition to a tandem duplication in the exon 3 (16
). The AR(H874Y) in CWR22Rv1 cells has been reported to be more sensitive to adrenal androgen dehydroepiandrosterone and anti-androgen hydroxyflutamide. Our work presented here characterizes a new androgen-dependent human prostate cancer cell line that expresses AR having the H874Y mutation without the tandem duplication in the exon 3. This is the first time such a human prostate cancer cell line is reported. CWR22Pc cells will provide a valuable experimental tool to investigate the importance of H874Y mutation in the AR for androgen regulation of prostate cancer cells as well as for development of androgen-independence.
Cytokine signaling pathways are active in CWR22Pc cells
One of the key molecular mechanisms that promote prostate cancer cell growth involves protein kinase growth factor signaling pathways. These cell signaling pathways regulate prostate cancer cell survival, proliferation and/or differentiation independently of AR or by affecting the transcriptional activity of AR. To investigate the activation status of the key known signaling pathways influencing growth and AR activity in prostate cancer cells, phosphorylation and total protein levels of Stat5a/b, Akt, MAPK and Stat3 (27
) were examined in exponentially growing CWR22Pc, CWR22Rv1, LNCaP and DU145 cells. Immunoprecipitation and immunoblotting of Stat5a/b shows that Stat5a/b is active in CWR22Pc cell line whereas Stat3 is constitutively active only in DU145 cells but not in CWR22Pc cells (, upper panel). The filters were stripped and re-blotted for total Stat5a/b and Stat3 protein levels. In addition, parallel samples of the cell lysates were immunoblotted with anti-actin to demonstrate the total protein level in each lane. Immunoblotting of whole cell lysates with anti-phospho-MAPK-p44/42 antibody showed that MAPK is activated at high level in exponentially growing CWR22Pc cells, while phosphorylated at a lower level in LNCaP, CWR22Rv1 and DU145 cells (). Furthermore, also Akt is phosphorylated in CWR22PC cells (). Parallel samples were immunoblotted to demonstrate total ERK and AKT levels as well as actin expression to demonstrate equal protein loading.
Protein kinase signaling pathways are activated in CWR22Pc cells
In the next set of experiments, we wanted to investigate the inducibility of Stat5a/b and Stat3 activation by cytokines prolactin (Prl) and interleukin-6 (IL-6) in CWR22Pc cells in comparison to LNCaP, CWR22Rv1 and DU145 cells. The cells were first grown in a regular growth medium containing 10% FBS, serum-starved over-night and stimulated with human Prl (10 nM) or IL-6 (4 nM) for 15 min. Stat5a, Stat5b () or Stat3 () were immunoprecipitated and blotted with antibodies recognizing phosphorylated Stat5a/b or Stat3, respectively. The filters were stripped and re-blotted with anti-Stat5ab or anti-Stat3 mAbs to demonstrate equal loading. Prl stimulation of CWR22Pc cells induced predominantly activation of Stat5b while the level of Stat5a expression in CWR22Pc cells was generally low. In CWR22Rv1 cell line, Stat5a and Stat5b were expressed at equal levels and Prl induced phosphorylation of both Stat5a and Stat5b in CWR22Rv1 cells, whereas Prl did not activate Stat5a/b in DU145 or LNCaP cells (). IL-6 stimulated phosphorylation of Stat3 in CWR22Pc cells. In addition, IL-6 induced activation of Stat3 in LNCaP and DU145 cells, but not in CWR22Rv1 cells. In conclusion, Stat5a/b and Stat3 in CWR22Pc cells are activated by Prl and IL-6, respectively.
The CWR22Pc cell line provides a useful research tool to address a number of key questions pertinent to the basic biology of prostate cancer as well as clinical management of the disease. First, since growth of CWR22Pc cells in culture is strictly regulated by androgens, CWR22Pc cells will be able to provide critical information about androgen-regulated growth mechanisms in a prostate-specific cell context. Second, the CWR22Pc cell line provides a model system where significance of different protein kinase signaling pathway activation can be tested for their ability to replace androgens for growth promotion and maintenance of prostate cancer cell viability in vitro and in vivo. Third, CWR22Pc cells have the H874Y mutation in the AR gene without additional genetic changes and will therefore enable studies of AR (H874Y) transcriptional activity in a biological setting where prostate-specific co-activators and co-repressors are expressed and present. Importantly, the re-growth of CWR22Pc tumors in vivo in nude mice after androgen deprivation mimics the course of development of hormone-refractory prostate cancer in humans. Future studies need to characterize genetically and epigenetically the cell clones that recur when CWR22Pc cells are grown as xenograft tumors under the biological pressure of androgen deprivation. Finally, it will be important to establish whether CWR22Pc cells metastasize when inoculated orthotopically in nude mice.