Currently, there is no universally effective therapy for the treatment of sarcoids which remains a major clinical challenge. The efficacy of different treatments is difficult to assess because most studies have not been designed to include controls and are frequently based on referral populations of horses treated at veterinary hospitals[5, 36, 37]. In the present study we have examined the efficacy of two DNA damaging agents to induce apoptosis.
Our data show that BPV-1 cells are more sensitive to the apoptotic effects of both UVB and cisplatin compared control cells. UVB reduces the half-life of E2; and we have shown that loss of BPV-1 E2 leads to increased apoptosis which may explain this finding. The BPV-1 mediated sensitisation to cisplatin may explain the relatively good success rates and low toxicity reported with cisplatin in equine sarcoids; Theon et al., demonstrated cisplatin to be effective over a two year period in 90% of cases[29, 39, 40]; Hewes and Sullins have reported an 85% success rate in equine sarcoids after a 2 year follow-up. Despite the increased apoptosis detected, BPV-1 cells are better able to survive the DNA insult compared to control cells. This is likely due to a small subset of resistant clones and the repeated use of cisplatin may therefore select for cells that are particularly resistant to apoptosis suggesting that cisplatin may not be appropriate for repeated treatment of sarcoids.
We have shown that p53 is overexpressed in BPV-1 cell lines. The p53 tumor suppressor protein plays a key role in coordinating cell cycle arrest, DNA repair, and programmed cell death following DNA damage. P53 protein is relatively unstable and has a short half-life, and is usually undetectable in normal cells by immunohistochemistry. In contrast, overexpression of p53 is often associated with the presence of mutant p53, which has a longer half life. However, there is no evidence to date for the presence of p53 gene mutations in equine sarcoids[43, 44] and our sequence analysis of EqS04b cells demonstrates wild type p53 (data not shown), although the possibly of p53 gene mutations in the other cell lines cannot be ruled out. Overexpression of wild type p53 has been observed in tumours[45, 46] and whilst the reason for this or its biological significance is not known, several studies have shown that it may be due to changes in the functionality of proteins that interact and control the activity and the levels of p53, such as MDM2 and MDMX. It has also been suggested that the overexpression may be a result of downregulation of ubiquitin mediated proteasome degradation. We show that the overexpression of wild type p53 in EqS04b cells is (at least in part) due to abnormal p53 stability however further studies are necessary to understand the mechanisms involved and the clinical significance. In HPV, p53 expression is stabilised by the E7 oncoprotein.
We show that in 2 sarcoid derived cell lines (EqS04b, EqS013) p53 overexpression is cytoplasmic. Cytoplasmic sequestration of p53 has been proposed as an important mechanism to disrupt its function as a tumor suppressor and has been reported in a range of tumours including breast cancers and neuroblastomas[51, 52]. Cytoplasmic sequestration of wild type correlates with attenuation of DNA damage-induced G1 arrest and apoptosis in neuroblastoma cell lines. Several factors are known to induce p53 cytoplasmic localisation including Jab1, which facilitates p53 nuclear exclusion and degradation and, Parc, a parkin-like ubiquitin ligase, has recently been shown to function as cytoplasmic anchor protein of p53. The pattern of cytoplasmic staining seen in this study using the D0-7 antibody in EqS04b cells is very different from the disperse uniform cytoplasmic staining of the cytoplasm we have previously observed with CM-1 antibody. In fact, in our previous study, CM-1 antibody showed cytoplasmic staining in EqS01a and S6-2 cells but not in control EqPalFs, however with the D0-7 antibody expression is clearly nuclear and expression is induced following UV exposure. These data suggest that CM-1 antibody and D0-7 antibody may recognise different forms of p53; additionally it appears that D0-7 antibody is best able to recognise equine p53[57, 58]. We and others have previously shown that a subset of equine sarcoid tumours express cytoplasmic p53[57–60] We have also shown that in an equine sarcoid derived cell line sarc-1 with cytoplasmic p53, no mRNA expression of the p53 target gene, mdm2 could be detected either before or after UV treatment showing that p53 transcriptional activation function is compromised in cells with cytoplasmic p53. The aberrant expression of p53 may also contribute to the increased clonogenic survival following DNA damage insult, but this speculative and remains to be established.
Our preliminary analysis of p53 expression and clinical type of sarcoid shows that more sarcoid aggressive sarcoids (fibroblastic and nodular) have higher levels of p53 positivity than quiescent verrucose sarcoids. However, the numbers of samples in this study are small and further studies are warranted to substantiate this initial finding.
In summary, we show that BPV-1 transformed equine fibroblasts are more sensitive to the apoptotic effects of cisplatin and UVB than control cells but show increased clonogenic survival. We show that in sarcoid cell lines, BPV-1 increases p53 expression within the nucleus but in some cell lines, p53 expression is cytoplasmic.