Browsing by Subject "Melanoma"
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- ItemOpen AccessThe detection of occult metastatic disease in patients with cutaneous melanoma(1999) Hanekom Gideon S; Kidson, SusanThe ability to identify melanoma patients with progressive disease is central to efficient management. The challenge therefore, is to develop prognostic markers and techniques which will allow the identification of those patients whom, at the time of primary tumor diagnosis, already have micrometastases (occult or clinically undetectable metastases). The use of the reverse transcription-PCR (RT-PCR) technique for the detection of circulating melanoma cells (CMCs) is potentially a powerful tool for identifying those patients at risk for developing metastases. The first aim of this study was to develop a more sensitive, reproducible, cost effective and clinically applicable assay and to eliminate the problem of false positives. A combined RT-PCR assay for tyrosinase mRNA, a marker specific for melanoma cells, was developed and tested. It was shown that the assay can reproducibly detect a single, viable melanoma cell in 10-15 ml of peripheral blood. Furthermore, a simple but effective procedure was developed to prevent carryover contamination. It was found that the chance of obtaining normal melanocyte contamination with the needle prick during blood sampling was only 2% and that illegitimate transcription does not contribute to sporadic false positives. The second aim of this study was to determine whether the early detection of CMCs is of any clinical value to monitor melanoma progression. Peripheral blood samples from 143 patients with primary melanoma (PM) were analysed by RT-PCR for the presence of tyrosinase mRNA. Seven percent (10/143) of the patients with PM had detectable CMCs. The percentage of PCR-positive patients was higher for stage II patients (9.0%) compared to stage I (5.3%) but the difference was not significant. A significantly higher percentage (P < 0.05) PCR-positive patients were found to have tumors greater than 1.5 mm thick and with ulceration present. Although this finding supports the notion that tumor thickness and ulceration are the two most significant prognostic factors, it was not possible at this stage, to link this directly to a poor prognosis since the majority of the PCR-positive patients have not yet (within four years) developed metastatic disease. However, the data does indicate that cells from tumors greater than 1.5 mm thick and with ulceration have a greater propensity to enter the circulation but that these cells do not necessarily have the ability to establish metastases. The results suggest that the detection rate of 9% for patients with stage II disease is much lower than would be expected, since 23.9% (16/67) of the stage II patients subsequently developed metastases. Of these 16 patients, only one was PCR-positive, one week before the metastases became clinically evident. Thus, the current technique fails to predict the likelihood of developing metastatic disease (P = 0.3485). The other nine PCR-positive patients had not yet developed metastases after a median follow-up period of four years. It is concluded that the current technique for the detection of CMCs is of limited clinical value to predict the likelihood of metastasis in patients with PM. It is suggested that other anatomic compartments, such as sentinel lymph nodes, should be explored for the identification of patients at risk for developing metastases. The third aim of this study was to determine whether high or low plasma levels and/or activity of plasminogen activator inhibitor type 1 (PAI1) correlate with the presence of metastatic disease in patients with melanoma. PAI1 is considered to be the main regulator of fibrinolytic activity in blood and has been identified as a key enzyme in the metastasis and vascularization of solid tumors. A unique enzyme-linked immunosorbant assay was developed to measure both the total amount of PAI1 in plasma as well as the active fraction of the inhibitor. This novel assay was then used to analyse and compare the plasmatic PAI1 levels and activity of a group of patients with advanced melanoma (AM) with a group of patients with primary disease and a control population. There was no statistical difference in the total plasmatic PAI1 levels between the controls and patients with PM and AM (P = 0.6199). In contrast, there was a significant difference in the active fraction of PAI1 between the controls and patients with PM or AM (P = 0.0076). A value of less than 44% active PAI1 was shown to be clinically meaningful by linear discriminant analysis. This means that a melanoma patient with a plasmatic PAI1 activity value less than 44% will have a 50% chance of harbouring metastases. Of the patients with PM, 19% had PAI1 activity values less than 44%, which strongly supports further investigations to determine whether plasmatic PAI1 activity levels might be predictive of metastatic disease. The false positive rate was 2.6%. It is speculated that this reduction in the active fraction of PAI1 for patients with AM might be attributed to tumor-derived tissue plasminogen activator and/or other melanoma-derived proteases or factors. The last section of this study describes several monoclonal antibodies (Mabs) that were developed against PAI1 in order to obtain useful reagents to study the regulatory functions of PAI1 in the metastasis and vascularization of solid tumors. The baculovirus expression system was used to express human PAI1 in insect cells and the crude infected cell population was used as the immunogen in mice. This approach was followed since the Escherichia coli-derived recombinant molecule elicited a poor immune response. A unique panel of anti-PAI1 Mabs was developed that were characterized with regard to their use for immunoblotting, immunofluorescence and immunocytochemistry. One of these antibodies blocked the binding of PAI1 to vitronectin and inhibited the activity of the inhibitor. Finally, two of these Mabs turned out to be extremely valuable and were used to develop a novel microtiter plate assay for measuring the active fraction of PAI1 in biological fluids by making use of Mabs against different epitopes of PAI1.
- ItemOpen AccessProteolytic mechanisms involved in the metastasis of human melanoma cells(1994) Fletcher, Jean Margaret; Dowdle, Eugene BThe metastatic process requires that tumour cells are capable of traversing various micro-environmental barriers, such as the basement membrane. There are various proteolytic mechanisms which could contribute to the process, plasminogen activation by tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) is one such mechanism. Extensive reports in the literature (reviewed in the introduction) indicate that most tumour cells synthesize uPA and that it is this enzyme, particularly when receptor-bound, which plays a role in invasion. UCT-Mel 3 is a human malignant melanoma cell line which was established in our laboratory, and has been shown to be highly metastatic in the nude mouse. This cell line is typical of many melanomas in that it synthesizes only tPA and not uPA. In part 1 of this thesis I further investigated the plasminogen activator production by these cells (at the level of mRNA as well as activity) as well as expression of plasminogen activator inhibitor PAl-1 and receptors for tPA and uPA (uPAR). UCT-Mel 3 cells expressed uPAR although uPA was not detected. I also examined cells cultured from two metastatic deposits. Interestingly, the metastatic cells produced PAl-1 which was undetected in the parent cells. After confirming that UCT-Mel 3 do not express detectable levels of uPA, I attempted (in part 2) to determine whether tPA could play a comparable role to that of uPA in the invasive process. My strategy was to inhibit the expression of tPA via two different methods, namely the use of antisense RNA and ribozyme. I then hoped to isolate clones producing no tPA, which would have been injected into nude mice in order to assay for metastasis. Unfortunately, neither of these methods proved to be successful in abrogating tPA expression. I was thus unable to achieve the ultimate aim of the project. However, during the course of the study a number of unforeseen problems arose. Firstly, the clonal variation within the cell population, and secondly, my inability to obtain antisense transfectants. I have speculated that a possible reason for the latter may be that the cells are in fact unable to grow in the absence of tPA.
- ItemOpen AccessThe T-box transcription factor, TBX3, is sufficient to promote melanoma formation and invasion(BioMed Central Ltd, 2013) Peres, Jade; Prince, SharonThe T-box transcription factor, TBX3, is overexpressed in several cancers and has been proposed as a chemotherapeutic target. Several lines of evidence suggest that TBX3 may be a key contributor to malignant melanoma, a highly aggressive and intractable disease. Using in vitro and in vivo assays we demonstrate here for the first time that overexpressing TBX3 in non-tumourigenic early stage melanoma cells is sufficient to promote tumour formation and invasion. Furthermore, we show that TBX3 may play an important role as a reciprocal switch between substrate dependent cell proliferation and tumour invasion.
- ItemOpen AccessThe protective role of DOT1L in UV-induced melanomagenesis(2018) Yin, Chengqian; Zhang, Jie; Wei, Wenyi; Wei, Zhi; Pan, Jingxuan; Wang, Yongjun; Xuan, Zhenyu; Hess, Jay; Hayward, Nicholas K; Goding, Colin R; Chen, Xiang; Zhou, Jun; Cui, RutaoThe DOT1L histone H3 lysine 79 (H3K79) methyltransferase plays an oncogenic role in MLL-rearranged leukemogenesis. Here, we demonstrate that, in contrast to MLL-rearranged leukemia, DOT1L plays a protective role in ultraviolet radiation (UVR)-induced melanoma development. Specifically, the DOT1L gene is located in a frequently deleted region and undergoes somatic mutation in human melanoma. Specific mutations functionally compromise DOT1L methyltransferase enzyme activity leading to reduced H3K79 methylation. Importantly, in the absence of DOT1L, UVR-induced DNA damage is inefficiently repaired, so that DOT1L loss promotes melanoma development in mice after exposure to UVR. Mechanistically, DOT1L facilitates DNA damage repair, with DOT1L-methylated H3K79 involvement in binding and recruiting XPC to the DNA damage site for nucleotide excision repair (NER). This study indicates that DOT1L plays a protective role in UVR-induced melanomagenesis.
- ItemOpen AccessUV-mediated Regulation of the anti-senescence factor Tbx2(2008) Abrahams, Amaal; Mowla, Shaheen; PARKER, M Iqbal; Goding, Colin R; Prince, SharonSeveral lines of evidence have implicated members of the developmentally important T-box gene family in cell cycle regulation and in cancer. Importantly, the highly related T-box factors Tbx2 and Tbx3 can suppress senescence through repressing the cyclin-dependent kinase inhibitors p19(ARF) and p21(WAF1/CIP1/SDII). Furthermore, Tbx2 is up-regulated in several cancers, including melanomas where it was shown to function as an anti-senescence factor, suggesting that this may be one of the mechanisms by which T-box proteins contribute to the oncogenic process. However, very little is known about whether Tbx2 is regulated by p21-mediated stress-induced senescence signaling pathways. In this study, using the MCF-7 breast cancer cell line known to overexpress Tbx2, we show that in response to stress induced by ultraviolet irradiation the Tbx2 protein is specifically phosphorylated by the p38 mitogen-activated protein kinase. Using site-directed mutagenesis and in vitro kinase assays, we have identified serine residues 336, 623, and 675 in the Tbx2 protein as the p38 target sites and show that these sites are phosphorylated in vivo. Importantly, we show by Western blotting, immunofluorescence, and reporter assays that this phosphorylation leads to increased Tbx2 protein levels, predominant nuclear localization of the protein, and an increase in the ability of Tbx2 to repress the p21(WAF1/CIP1/SDII) promoter. These results show for the first time that the ability of Tbx2 to repress the p21 gene is enhanced in response to a stress-induced senescence pathway, which leads to a better understanding of the regulation of the anti-senescence function of Tbx2.