I am currently carrying out a project examining the potential effect HPV has on keratin expression in HeLa cells and C33a's which will be divided into those transfected with a HPV plasmid, and those not including the plasmid. I'm finding it difficult to pinpoint the markers used for keratin expression, would someone be able to point me in the right direction. I understand a panel may be necessary, but which immunohistochemical markers for cytokeratin would be the main ones to use here?
I have no idea if HPV has an effect on keratin expression - I wouldn't think so, but HPV likes to infect reserve cells. These reserve cells can still differentiate between squamous and glandular epithelium. If the cervical cancer is a squamous cell ectocervical carcinoma, p63, CK 14, and CK 5/6 will be positive - not because of HPV, but because of their squamous nature. If the cervical cancer arises in the glandular endocervical epithelium, the normal glandular markers will be positive: CAM 5.2, CK7, CK18/19. Of note, CK7 sometimes stains squamous epithelium in the cervix as well.
So I would advice to stain with a panel of cytokeratin markers and see if there's up- or downregulation. Maybe you can also use EMT or MET markers (epithelial-to mesenchymal transition) ?
Ii assume squamous cell carcinoma of the cervix. In that case high molecular cytokeratins such as AE1/AE3; Kermix, MAK6 would be positive, as well as ck5/6 and p63 which are rather specific for squamous differentiation. Low molecular weight cytokeratins such as CK8/18 would be negative. Now, this immunopprofile is useful for well differentiated carcinomas. My understanding is that HeLa is a poorly differentiated cell line.
We did some research on CK7 and CK17 expression in cervical precancer. Especially CK17 seemed promising.
F Smedts et al published about CK markers, e.g.: Expression of keratins 1, 6, 15, 16, and 20 in normal cervical epithelium, squamous metaplasia, cervical intraepithelial neoplasia, and cervical carcinoma.
Smedts F1, Ramaekers F, Leube RE, Keijser K, Link M, Vooijs P.
The endocervical reserve cell has been shown to express keratins 5, 8, 14, 17, 18, and 19.So it may be a good idea to use a panel of the above cytokeratins and as far as I know HPV targets these reserve cells.
To really understand effects of HPV on keratins, you will need to include a broad panel that reflects both reserve cell and squamous cell keratins. I would include 5, 7, 8, 14, 17, 18 and 19. I would not include k1 or k10, since squamous cells do not differentiate significantly in monolayer culture. I don't understand why you want to look at HeLa cells, since they already contain HPV 18 and express the viral E6 and E7 genes.
If the question is about cytokeratin markers in cervical cancer, than from the clinical point of view, the one is cytokeratin fragment 19 (CYFRA 21.1). The subject of HPV and all around it in cervical cancer is very complicated, but since cytokeratins are considered try the one.
p63 and CK5/6 in squampus cell carcinoma. CK7 and/or cam 5.2 in endocervical adenocarcinoma.
Ck 19 seems to me the most promissing cytokeratin. I'm not sure you can sepparate hpv action from squamous cell nature cytokeratin expression. Good luck.
p16 expression can also help as it is a surrogate marker for HPV infection.
There can be higher expression of CKs 10, 14, 17, 18, 19 in planoepithelial cervical cancer.
Thanks guys, I'm going to use a pan cytokeratin- Ae1-Ae3 to begin with. I'm transforming a plasmid containing the HPV 16 genes into Bl21 cells, and then transvecting them into the c33a's.
He La cells have HPV already? Just check this...I remember coming across a paper somewhere..may affect your results
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