Whilst your comments are relevant, I would recommend against getting a sample from another researcher, as the same problems may occur. If you can get another vial from ATCC or DSMZ, then this is a viable option, but you may have performed a lot of lengthy experiments on the cells you have already. Therefore acquiring additional cells may not be tenable, or at least could result in a huge amount of additional work to characterize your new cells for whatever phenotype you are studying. If this is the case, then the primary focus will be to establish the origin of the cells you are working on.

Furthermore, your point about clonal evolution is valid, but is it really an issue? PC3 has an incredibly rearranged genome and that level of rearrangement is almost never seem in the context of primary prostate cancer. The cell line was derived from an aggressive tumour and has been established for many years. In general, cancer cell lines by definition have acquired many properties (such as DNA and chromosomal lesions) that provide an in vitro growth advantage and the genome will have evolved significantly since the cell line was established. There is the long-standing question of how accurately cell lines reflect human disease? Any given cell line may have an important biological/ cellular phenotype worthy of study, but whether that cell line reflects the pathogenesis of the derived tumour is in question.

Therefore, the biological properties of a cell line are likely to the critical research issue, so you have to wonder how relevant any degree of clonal evolution is, particularly in the context of a system defined by genomic instability and clonal evolution. That said, this is a long standing and contentious question, and many individuals will have different and equally relevant views on the subject.

However, even if you might consider clonal evolution to be relevant, it is highly unlikely that STR analysis (at least in the context of the commercial assays available) will provide meaningful insight into clonal evolution. Depending on the level of contamination, the same could be said for the use of STR analysis in this context.

Cytogenetics or molecular cytogenetics may not be appropriate in all situations, but to suggest that it cannot measure chromosomal heterogeneity between cells is incorrect. After all, it is a single cell approach (with the exception of CGH approaches). Of course, there are issues of sensitivity, particularly in light of modern technologies, but one of the premises of the clinical application of cytogenetics has been to identify genetic heterogeneity, in the context of cancer and constitutional disease. Examples would be mosaic aneuploidy, maternal contamination of prenatal samples, and clonal evolution of haematological malignancies. The entire purpose of the development of Hooks tables was to exclude levels of chromosomal mosaicism.

Of course, to exclude low levels of contamination or subtle clonal evolution would be beyond the realistic limits of cytogenetic or molecular cytogenetic approaches, even with the design of informative FISH probes for interphase analysis.

Thank you Gaurav. I haven't touched HeLas so there should be no danger of that, and morphologically they look identical to when I started.

I wonder if you, or anyone, knows of PC-3 specific markers I should be looking for?

Hi Matthew

The best way to ensure your cell lines are really what they say on the label is to STR genotype them. This is recommended by the NIH and is (apparently) going to be required by major journals for publication.

We STR genotype 'in house' at our institute, but you can easily buy a kit for it, or outsource it to one of the myriad of service providers (you just need to send them some DNA usually). Our biorepository staff recommend STR genotyping and the beginning and end of every large project in the lab (although I feel this is overkill).

Basically the STR assay will give you a 'probability' that your cell line is what you think it is. It compares PCR generated STR profiles with those held at ATCC or ECACC (I think), and as long as the cell lines are in their databases you'll get a percent identity score. There is an assay for human cell lines, and also a less well characterised one for mouse cell lines. Most common human cell lines (eg PC3 cells) will be in the databases. Also, just for completeness I should mention that the human authentication assay comes in two different PCR primer collections.

Hope this helps.

Al

Alasdair is correct. Most journals now require the STR genotyping to be done before they will publish your work. Unless you can prove the cells were bought in the last two years and maintained between certain passages for experimentation this will need done to publish. Sometimes its better just to get a new batch of cells from DSMZ or ECACC £300 as STR can nearly cost as much £200 sometimes!

PC-3 cells are do not have androgen receptor so check the expression of androgen receptor or simply you can check the effect of testosterone treatment on the growth characteristics. However, for confirmatory assessment you have to do much more complicated study which is not required. I mean instead to doing such complicated experiments you can get a new vial of cells from ATCC.

As Alasdair says, more and more journals are asking for STR genotyping. I am not sure of the cost but believe is not cheap. We have started replacing cell lines regularily, bought from ATCC, ECACC and DSMZ; and do not passage more than 10 times. So far we haven't had any problems with publications or queries with regards to the cell lines.

Same as Alasdair is saying STR genotyping is one of the best tools to confirm your cell line is actually the one you think it is. I recently had the chance of hearing a very interesting webinar in which they mention the work of Amanda Capes-Davis and Ian Freshney, they developed a database of known misidentified or contaminated cell lines available here: http://standards.atcc.org/kwspub/home/the_international_cell_line_authentication_committee-iclac_/Database_of_Cross_Contaminated_or_Misidentified_Cell_Lines.pdf Hope it helps you and check out more about the International Cell Line Authentication Committee (ICLAC).

Whilst there are journal requirements for cell line validation through STR analysis, karyotyping or MFISH analysis would unquestionably confirm that your cell line is PC3. The genome is absolutely unique by both techniques and has been published by several groups. See Cancer genetics and Cytogenetics. 01/2001; 124(2):112-21. I only mention this because you may be able to access these technologies more readily.

If you have concerns that your cell line might not be PC-3, order a new vial from ATCC or DSMZ or write to a researcher asking for a sample. Cytogenetics will not exclude the possibility of cross-contamination with another cell line or clonal evolution in culture.

Whilst your comments are relevant, I would recommend against getting a sample from another researcher, as the same problems may occur. If you can get another vial from ATCC or DSMZ, then this is a viable option, but you may have performed a lot of lengthy experiments on the cells you have already. Therefore acquiring additional cells may not be tenable, or at least could result in a huge amount of additional work to characterize your new cells for whatever phenotype you are studying. If this is the case, then the primary focus will be to establish the origin of the cells you are working on.

Furthermore, your point about clonal evolution is valid, but is it really an issue? PC3 has an incredibly rearranged genome and that level of rearrangement is almost never seem in the context of primary prostate cancer. The cell line was derived from an aggressive tumour and has been established for many years. In general, cancer cell lines by definition have acquired many properties (such as DNA and chromosomal lesions) that provide an in vitro growth advantage and the genome will have evolved significantly since the cell line was established. There is the long-standing question of how accurately cell lines reflect human disease? Any given cell line may have an important biological/ cellular phenotype worthy of study, but whether that cell line reflects the pathogenesis of the derived tumour is in question.

Therefore, the biological properties of a cell line are likely to the critical research issue, so you have to wonder how relevant any degree of clonal evolution is, particularly in the context of a system defined by genomic instability and clonal evolution. That said, this is a long standing and contentious question, and many individuals will have different and equally relevant views on the subject.

However, even if you might consider clonal evolution to be relevant, it is highly unlikely that STR analysis (at least in the context of the commercial assays available) will provide meaningful insight into clonal evolution. Depending on the level of contamination, the same could be said for the use of STR analysis in this context.

Cytogenetics or molecular cytogenetics may not be appropriate in all situations, but to suggest that it cannot measure chromosomal heterogeneity between cells is incorrect. After all, it is a single cell approach (with the exception of CGH approaches). Of course, there are issues of sensitivity, particularly in light of modern technologies, but one of the premises of the clinical application of cytogenetics has been to identify genetic heterogeneity, in the context of cancer and constitutional disease. Examples would be mosaic aneuploidy, maternal contamination of prenatal samples, and clonal evolution of haematological malignancies. The entire purpose of the development of Hooks tables was to exclude levels of chromosomal mosaicism.

Of course, to exclude low levels of contamination or subtle clonal evolution would be beyond the realistic limits of cytogenetic or molecular cytogenetic approaches, even with the design of informative FISH probes for interphase analysis.

Jon, I agree with the points you raise. That is exactly why one would specify the request: For example

Dear Dr. Garrett,

I have read your recent paper in Clin Cancer Res. describing the antitumor activity of the novel clinical drug candidate AT13148, with great interest. I am writing to request one of the aliquots from your PC3 cell line master or working cell bank established from the original vial you received from ATCC. This would help us in our academic research immensely as we recently became aware of a possible cross-contamination in our PC3 cultures.

Thank you very much for your consideration and assistance in this matter. I look forward to receiving your MTA and other applicable documents.

Kind regards...

Wow. Thanks everybody.. It never ceases to amaze me how helpful people are on this forum.

I'm going to consider all the options, but thankfully, having kept the cells in HAMS F12, only ever going as far as passage 30, and looking at their morphology daily, I am very confident they are what they say they are!

Will repost when I have a definite result either way..! Thanks again