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Proteome serological determination of tumor-associated antigens in melanoma.

Forgber M, Trefzer U, Sterry W, Walden P - PLoS ONE (2009)

Bottom Line: One of these antigens, galectin-3, has been related to various oncogenic processes including metastasis formation and invasiveness.Similarly, enolase has been found deregulated in different cancers.With at least 2 of 18 identified proteins implicated in oncogenic processes, the work confirms the potential of proteome-based antigen discovery to identify pathologically relevant proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Venerology and Allergy, Charité Universitätsmedizin Berlin, Humboldt University, Berlin, Germany.

ABSTRACT
Proteome serology may complement expression library-based approaches as strategy utilizing the patients' immune responses for the identification pathogenesis factors and potential targets for therapy and markers for diagnosis. Melanoma is a relatively immunogenic tumor and antigens recognized by melanoma-specific T cells have been extensively studied. The specificities of antibody responses to this malignancy have been analyzed to some extent by molecular genetic but not proteomics approaches. We screened sera of 94 melanoma patients for anti-melanoma reactivity and detected seropositivity in two-thirds of the patients with 2-6 antigens per case detected by 1D and an average of 2.3 per case by 2D Western blot analysis. For identification, antigen spots in Western blots were aligned with proteins in 2-DE and analyzed by mass spectrometry. 18 antigens were identified, 17 of which for the first time for melanoma. One of these antigens, galectin-3, has been related to various oncogenic processes including metastasis formation and invasiveness. Similarly, enolase has been found deregulated in different cancers. With at least 2 of 18 identified proteins implicated in oncogenic processes, the work confirms the potential of proteome-based antigen discovery to identify pathologically relevant proteins.

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Related in: MedlinePlus

Pattern of seroreactivities of melanoma patients, healthy controls and patients with other diseases against the melanoma cell line M-NRT.Total protein extract of the tumor cells were separated by SDS-PAGE, blotted onto nitrocellulose and probed with the sera of the melanoma patients (Panels A–C), of healthy control donors (Panel D, sera 102–110) or patients with cutaneous lymphoma (Panel E, sera 96–98), pancreas carcinoma (Panel E, serum 101) or visceral leishmaniasis (Panel E, sera 111–113). The numbers atop of each lane represent the number of the sera and are used throughout this report. For comparison, melanoma serum 7 was included in all blots. The Western blot analyses were done with the sera at a dilution of 1/6 in a multiple channel blotting/Western blot developing chamber for panels A–E. The letters underneath the lanes indicated the combinations of sera for the multiple probing of the 2D Western blots shown in Figure 2 and summarized in Table 1. Panel F: High-resolution 1D Western blot for comparison of the seroreactivities of melanoma patients and a healthy donor against the melanoma cell line M-NRT. In contrast to the blots shown in panels A–E, the sera were applied 1/200 to isolated lanes from SDS-PAGE in sealed plastic bags which results in a better definition of the bands when compared to blots from multiple blotting devices. Serum 18 is autologous to tumor cell line M-NRT, serum 7 is from a different patient and serum 103 from a healthy donor. The arrows to the left of the lanes indicated antigen bands that are shared between the patients and the healthy controls and occur in every blot. The arrows to the right of the lanes indicated prominent antigen that are detected only by the sera of melanoma patients.
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pone-0005199-g001: Pattern of seroreactivities of melanoma patients, healthy controls and patients with other diseases against the melanoma cell line M-NRT.Total protein extract of the tumor cells were separated by SDS-PAGE, blotted onto nitrocellulose and probed with the sera of the melanoma patients (Panels A–C), of healthy control donors (Panel D, sera 102–110) or patients with cutaneous lymphoma (Panel E, sera 96–98), pancreas carcinoma (Panel E, serum 101) or visceral leishmaniasis (Panel E, sera 111–113). The numbers atop of each lane represent the number of the sera and are used throughout this report. For comparison, melanoma serum 7 was included in all blots. The Western blot analyses were done with the sera at a dilution of 1/6 in a multiple channel blotting/Western blot developing chamber for panels A–E. The letters underneath the lanes indicated the combinations of sera for the multiple probing of the 2D Western blots shown in Figure 2 and summarized in Table 1. Panel F: High-resolution 1D Western blot for comparison of the seroreactivities of melanoma patients and a healthy donor against the melanoma cell line M-NRT. In contrast to the blots shown in panels A–E, the sera were applied 1/200 to isolated lanes from SDS-PAGE in sealed plastic bags which results in a better definition of the bands when compared to blots from multiple blotting devices. Serum 18 is autologous to tumor cell line M-NRT, serum 7 is from a different patient and serum 103 from a healthy donor. The arrows to the left of the lanes indicated antigen bands that are shared between the patients and the healthy controls and occur in every blot. The arrows to the right of the lanes indicated prominent antigen that are detected only by the sera of melanoma patients.

Mentions: To determine the frequencies and specificity patterns of antibody responses to melanoma-associated antigens, we tested by Western blot analyses the reactivities of sera of 94 patients with melanoma (see Table S1 for clinical details of the cases) against protein extracts from the melanoma cell line M-NRT separated by 1-dimensional SDS-PAGE (Figure 1A–C). As controls, the sera of 9 healthy individuals (Figure 1D), and of patients with cutaneous lymphoma, pancreas carcinoma or visceral leishmaniasis, an infectious disease that is known to induce responses to autoantigens of the infected host, were tested (Figure 1E). Overall, the signals were relatively weak given the high serum concentrations of 1∶6 used for these Western blots. A large number of faint background bands were detected with the sera of the healthy controls and patients alike as illustrated with Figure 1F which shows the Western blots with serum of a healthy donor (Serum 103) and the sera of two melanoma patients (Serum numbers 7 and 18). The Western blots shown in Figure 1F were developed as individual strips in separate plastic bags at a dilution of 1 in 200 which results in a better definition of the bands but is not suited for comparative screening of large numbers of sera. The arrows to the left of the Western blot lanes indicate shared signals found with patient and control sera alike. The arrows to the right of the lanes are stronger and indicate unique bands seen only with sera of melanoma patients. Serum 18 was from the same patient from whose tumor the melanoma cell line M-NRT had been established. This Western blot, thus, documents the reactivity in the autologous combination of tumor cells and serum. All other sera were from different patients or healthy donors, thus displaying the reactivity in heterogeneous combination of tumor cells and serum. The antigens thus detected are, therefore, expected to be antigens shared between the tumors of the serum donors and the test tumor cells M-NRT. With nearly two-thirds of the sera of the melanoma patients prominent bands are detectable that are not found with the healthy control sera or that are much stronger than those in the controls (Figure 1). The numbers of such prominent antigens detected with the reactive sera ranged between 2 and 6 per patient serum. Their masses were between 21 and 90 kDa with the bulk of the stronger bands between 40 and 80 kDa. The patients whose sera were tested represent, with the exception of ocular melanoma, all forms of melanoma. The majority of the patients were at stage 3 or 4 of disease but some were at earlier stages (Table S1). The course of disease ranged from relatively slow progression to aggressive disease. Within this range of patients there is no correlation of the numbers of antigens targeted, and frequencies or pattern of seropositivity with the clinical conditions. A few stronger reactivities were seen with the sera of healthy controls and patients with lymphoma, pancreas carcinoma or Leishmaniasis. These antigens were different than those detected with the sera of melanoma patients. In summary, about two-thirds of the patients had developed prominently detectable antibody responses against melanoma-associated antigens. The serospecificity patterns, however, were heterogeneous with no antigen that induced responses in a majority of the patients.


Proteome serological determination of tumor-associated antigens in melanoma.

Forgber M, Trefzer U, Sterry W, Walden P - PLoS ONE (2009)

Pattern of seroreactivities of melanoma patients, healthy controls and patients with other diseases against the melanoma cell line M-NRT.Total protein extract of the tumor cells were separated by SDS-PAGE, blotted onto nitrocellulose and probed with the sera of the melanoma patients (Panels A–C), of healthy control donors (Panel D, sera 102–110) or patients with cutaneous lymphoma (Panel E, sera 96–98), pancreas carcinoma (Panel E, serum 101) or visceral leishmaniasis (Panel E, sera 111–113). The numbers atop of each lane represent the number of the sera and are used throughout this report. For comparison, melanoma serum 7 was included in all blots. The Western blot analyses were done with the sera at a dilution of 1/6 in a multiple channel blotting/Western blot developing chamber for panels A–E. The letters underneath the lanes indicated the combinations of sera for the multiple probing of the 2D Western blots shown in Figure 2 and summarized in Table 1. Panel F: High-resolution 1D Western blot for comparison of the seroreactivities of melanoma patients and a healthy donor against the melanoma cell line M-NRT. In contrast to the blots shown in panels A–E, the sera were applied 1/200 to isolated lanes from SDS-PAGE in sealed plastic bags which results in a better definition of the bands when compared to blots from multiple blotting devices. Serum 18 is autologous to tumor cell line M-NRT, serum 7 is from a different patient and serum 103 from a healthy donor. The arrows to the left of the lanes indicated antigen bands that are shared between the patients and the healthy controls and occur in every blot. The arrows to the right of the lanes indicated prominent antigen that are detected only by the sera of melanoma patients.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667248&req=5

pone-0005199-g001: Pattern of seroreactivities of melanoma patients, healthy controls and patients with other diseases against the melanoma cell line M-NRT.Total protein extract of the tumor cells were separated by SDS-PAGE, blotted onto nitrocellulose and probed with the sera of the melanoma patients (Panels A–C), of healthy control donors (Panel D, sera 102–110) or patients with cutaneous lymphoma (Panel E, sera 96–98), pancreas carcinoma (Panel E, serum 101) or visceral leishmaniasis (Panel E, sera 111–113). The numbers atop of each lane represent the number of the sera and are used throughout this report. For comparison, melanoma serum 7 was included in all blots. The Western blot analyses were done with the sera at a dilution of 1/6 in a multiple channel blotting/Western blot developing chamber for panels A–E. The letters underneath the lanes indicated the combinations of sera for the multiple probing of the 2D Western blots shown in Figure 2 and summarized in Table 1. Panel F: High-resolution 1D Western blot for comparison of the seroreactivities of melanoma patients and a healthy donor against the melanoma cell line M-NRT. In contrast to the blots shown in panels A–E, the sera were applied 1/200 to isolated lanes from SDS-PAGE in sealed plastic bags which results in a better definition of the bands when compared to blots from multiple blotting devices. Serum 18 is autologous to tumor cell line M-NRT, serum 7 is from a different patient and serum 103 from a healthy donor. The arrows to the left of the lanes indicated antigen bands that are shared between the patients and the healthy controls and occur in every blot. The arrows to the right of the lanes indicated prominent antigen that are detected only by the sera of melanoma patients.
Mentions: To determine the frequencies and specificity patterns of antibody responses to melanoma-associated antigens, we tested by Western blot analyses the reactivities of sera of 94 patients with melanoma (see Table S1 for clinical details of the cases) against protein extracts from the melanoma cell line M-NRT separated by 1-dimensional SDS-PAGE (Figure 1A–C). As controls, the sera of 9 healthy individuals (Figure 1D), and of patients with cutaneous lymphoma, pancreas carcinoma or visceral leishmaniasis, an infectious disease that is known to induce responses to autoantigens of the infected host, were tested (Figure 1E). Overall, the signals were relatively weak given the high serum concentrations of 1∶6 used for these Western blots. A large number of faint background bands were detected with the sera of the healthy controls and patients alike as illustrated with Figure 1F which shows the Western blots with serum of a healthy donor (Serum 103) and the sera of two melanoma patients (Serum numbers 7 and 18). The Western blots shown in Figure 1F were developed as individual strips in separate plastic bags at a dilution of 1 in 200 which results in a better definition of the bands but is not suited for comparative screening of large numbers of sera. The arrows to the left of the Western blot lanes indicate shared signals found with patient and control sera alike. The arrows to the right of the lanes are stronger and indicate unique bands seen only with sera of melanoma patients. Serum 18 was from the same patient from whose tumor the melanoma cell line M-NRT had been established. This Western blot, thus, documents the reactivity in the autologous combination of tumor cells and serum. All other sera were from different patients or healthy donors, thus displaying the reactivity in heterogeneous combination of tumor cells and serum. The antigens thus detected are, therefore, expected to be antigens shared between the tumors of the serum donors and the test tumor cells M-NRT. With nearly two-thirds of the sera of the melanoma patients prominent bands are detectable that are not found with the healthy control sera or that are much stronger than those in the controls (Figure 1). The numbers of such prominent antigens detected with the reactive sera ranged between 2 and 6 per patient serum. Their masses were between 21 and 90 kDa with the bulk of the stronger bands between 40 and 80 kDa. The patients whose sera were tested represent, with the exception of ocular melanoma, all forms of melanoma. The majority of the patients were at stage 3 or 4 of disease but some were at earlier stages (Table S1). The course of disease ranged from relatively slow progression to aggressive disease. Within this range of patients there is no correlation of the numbers of antigens targeted, and frequencies or pattern of seropositivity with the clinical conditions. A few stronger reactivities were seen with the sera of healthy controls and patients with lymphoma, pancreas carcinoma or Leishmaniasis. These antigens were different than those detected with the sera of melanoma patients. In summary, about two-thirds of the patients had developed prominently detectable antibody responses against melanoma-associated antigens. The serospecificity patterns, however, were heterogeneous with no antigen that induced responses in a majority of the patients.

Bottom Line: One of these antigens, galectin-3, has been related to various oncogenic processes including metastasis formation and invasiveness.Similarly, enolase has been found deregulated in different cancers.With at least 2 of 18 identified proteins implicated in oncogenic processes, the work confirms the potential of proteome-based antigen discovery to identify pathologically relevant proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Venerology and Allergy, Charité Universitätsmedizin Berlin, Humboldt University, Berlin, Germany.

ABSTRACT
Proteome serology may complement expression library-based approaches as strategy utilizing the patients' immune responses for the identification pathogenesis factors and potential targets for therapy and markers for diagnosis. Melanoma is a relatively immunogenic tumor and antigens recognized by melanoma-specific T cells have been extensively studied. The specificities of antibody responses to this malignancy have been analyzed to some extent by molecular genetic but not proteomics approaches. We screened sera of 94 melanoma patients for anti-melanoma reactivity and detected seropositivity in two-thirds of the patients with 2-6 antigens per case detected by 1D and an average of 2.3 per case by 2D Western blot analysis. For identification, antigen spots in Western blots were aligned with proteins in 2-DE and analyzed by mass spectrometry. 18 antigens were identified, 17 of which for the first time for melanoma. One of these antigens, galectin-3, has been related to various oncogenic processes including metastasis formation and invasiveness. Similarly, enolase has been found deregulated in different cancers. With at least 2 of 18 identified proteins implicated in oncogenic processes, the work confirms the potential of proteome-based antigen discovery to identify pathologically relevant proteins.

Show MeSH
Related in: MedlinePlus