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Phorbol myristate acetate and Bryostatin 1 rescue IFN-gamma inducibility of MHC class II molecules in LS1034 colorectal carcinoma cell line.

Kudinov Y, Wiseman CL, Kharazi AI - Cancer Cell Int. (2003)

Bottom Line: The effect of PMA was not observed in two other poorly responding cell lines, MSTO-211H mesothelioma and HepG2 hepatocellular carcinoma, and was abrogated by relatively high concentrations of PKC inhibitors staurosporine (100 nM) and GF 109203X (1,000 nM).Both surface and intracellular staining of all cell lines with antibodies against IFNgR1 and IFNgR2 failed to detect any increase in IFNg receptor expression following incubation with PMA.In this study we showed that IFNg-inducibility of MHCII antigens in weakly inducible LS1034 colorectal carcinoma cell line can be rescued by concomitant incubation with PKC agonists.

View Article: PubMed Central - HTML - PubMed

Affiliation: Immunotherapy Laboratory, St,Vincent Medical Center, Los Angeles, CA, USA. YuriKudinov@DOCHS.org

ABSTRACT

Background: The expression of major histocompatibility complex class II (MHCII) antigens in both mouse and human tumors is rare, and these antigens are not easily inducible by IFN-gamma (IFNg). Since MHCII may play an important role in the development of host antitumor immune response, we explored the possibility of restoring MHCII inducibility in several IFNg-resistant tumor cell lines using protein kinase C (PKC) agonists phorbol myristate acetate (PMA) or Bryostatin.

Results: Tumor cells were co-cultured with various concentrations of PMA and IFNg for 48 hr. The expression of MHCII antigens and receptors IFNgR1 and IFNgR2 was determined by flow cytometry. We showed that the presence of as little as 0.1 ng/ml of PMA in tissue culture restored the ability of weakly inducible LS1034 colon carcinoma cells to express MHCII in response to IFNg (100 - 10,000 IU/ml) in a dose-dependent manner. Likewise, Bryostatin 1, as low as 10 ng/ml produced a 5-6 fold upregulation of MHCII. The effect of PMA was not observed in two other poorly responding cell lines, MSTO-211H mesothelioma and HepG2 hepatocellular carcinoma, and was abrogated by relatively high concentrations of PKC inhibitors staurosporine (100 nM) and GF 109203X (1,000 nM). Both surface and intracellular staining of all cell lines with antibodies against IFNgR1 and IFNgR2 failed to detect any increase in IFNg receptor expression following incubation with PMA.

Conclusion: In this study we showed that IFNg-inducibility of MHCII antigens in weakly inducible LS1034 colorectal carcinoma cell line can be rescued by concomitant incubation with PKC agonists. Bryostatin 1 may be considered for further investigation of IFNg-dependent MHCII induction in resistant tumors in vivo.

No MeSH data available.


Related in: MedlinePlus

PMA rescues MHCII inducibility in low responding LS1034 colon carcinoma cell line. Expression levels of MHCII (mean ± sd) induced by combined treatment with IFNg and PMA are plotted on the left (A) and expression levels of MHCII induced by combined treatment with IFNg, PMA and ethanol are plotted on the right (B). The experiment was repeated 4 times. Different treatments were compared to group 20a or 20b (see Table 1 for details on group codes). Asterisks indicate significant differences on post-hoc tests: *P < 0.05 by Tukey's HSD test; **P < 0.05 by Scheffe's test (see the supplementary file "ANOVA.xls" for original data used to perform this analysis).
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Figure 2: PMA rescues MHCII inducibility in low responding LS1034 colon carcinoma cell line. Expression levels of MHCII (mean ± sd) induced by combined treatment with IFNg and PMA are plotted on the left (A) and expression levels of MHCII induced by combined treatment with IFNg, PMA and ethanol are plotted on the right (B). The experiment was repeated 4 times. Different treatments were compared to group 20a or 20b (see Table 1 for details on group codes). Asterisks indicate significant differences on post-hoc tests: *P < 0.05 by Tukey's HSD test; **P < 0.05 by Scheffe's test (see the supplementary file "ANOVA.xls" for original data used to perform this analysis).

Mentions: The results demonstrated a substantial increase in MHCII expression in LS1034 cell line following combined incubation with PMA and IFNg (Figure 2). Two-factor analysis of variance revealed that the magnitude of MHCII induction in LS1034 cells was almost totally determined by concentration of IFNg (F2;36 = 29.3, P < 10-6). Higher response to IFNg tended to be associated with higher concentration of PMA in some experiments, but the overall effect of PMA did not reach a commonly accepted level of significance (F3;36 = 1.9, P = 0.14). Interestingly, the effect of PMA did reach significance in cultures supplemented with 172 mM ethanol (F3;36 = 3.0, P = 0.043; groups 09b→12b, 15b→18b, 21b→24b). No interaction effect between PMA and IFNg was found (F6;36 = 0.14, P = 0.99).


Phorbol myristate acetate and Bryostatin 1 rescue IFN-gamma inducibility of MHC class II molecules in LS1034 colorectal carcinoma cell line.

Kudinov Y, Wiseman CL, Kharazi AI - Cancer Cell Int. (2003)

PMA rescues MHCII inducibility in low responding LS1034 colon carcinoma cell line. Expression levels of MHCII (mean ± sd) induced by combined treatment with IFNg and PMA are plotted on the left (A) and expression levels of MHCII induced by combined treatment with IFNg, PMA and ethanol are plotted on the right (B). The experiment was repeated 4 times. Different treatments were compared to group 20a or 20b (see Table 1 for details on group codes). Asterisks indicate significant differences on post-hoc tests: *P < 0.05 by Tukey's HSD test; **P < 0.05 by Scheffe's test (see the supplementary file "ANOVA.xls" for original data used to perform this analysis).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC153529&req=5

Figure 2: PMA rescues MHCII inducibility in low responding LS1034 colon carcinoma cell line. Expression levels of MHCII (mean ± sd) induced by combined treatment with IFNg and PMA are plotted on the left (A) and expression levels of MHCII induced by combined treatment with IFNg, PMA and ethanol are plotted on the right (B). The experiment was repeated 4 times. Different treatments were compared to group 20a or 20b (see Table 1 for details on group codes). Asterisks indicate significant differences on post-hoc tests: *P < 0.05 by Tukey's HSD test; **P < 0.05 by Scheffe's test (see the supplementary file "ANOVA.xls" for original data used to perform this analysis).
Mentions: The results demonstrated a substantial increase in MHCII expression in LS1034 cell line following combined incubation with PMA and IFNg (Figure 2). Two-factor analysis of variance revealed that the magnitude of MHCII induction in LS1034 cells was almost totally determined by concentration of IFNg (F2;36 = 29.3, P < 10-6). Higher response to IFNg tended to be associated with higher concentration of PMA in some experiments, but the overall effect of PMA did not reach a commonly accepted level of significance (F3;36 = 1.9, P = 0.14). Interestingly, the effect of PMA did reach significance in cultures supplemented with 172 mM ethanol (F3;36 = 3.0, P = 0.043; groups 09b→12b, 15b→18b, 21b→24b). No interaction effect between PMA and IFNg was found (F6;36 = 0.14, P = 0.99).

Bottom Line: The effect of PMA was not observed in two other poorly responding cell lines, MSTO-211H mesothelioma and HepG2 hepatocellular carcinoma, and was abrogated by relatively high concentrations of PKC inhibitors staurosporine (100 nM) and GF 109203X (1,000 nM).Both surface and intracellular staining of all cell lines with antibodies against IFNgR1 and IFNgR2 failed to detect any increase in IFNg receptor expression following incubation with PMA.In this study we showed that IFNg-inducibility of MHCII antigens in weakly inducible LS1034 colorectal carcinoma cell line can be rescued by concomitant incubation with PKC agonists.

View Article: PubMed Central - HTML - PubMed

Affiliation: Immunotherapy Laboratory, St,Vincent Medical Center, Los Angeles, CA, USA. YuriKudinov@DOCHS.org

ABSTRACT

Background: The expression of major histocompatibility complex class II (MHCII) antigens in both mouse and human tumors is rare, and these antigens are not easily inducible by IFN-gamma (IFNg). Since MHCII may play an important role in the development of host antitumor immune response, we explored the possibility of restoring MHCII inducibility in several IFNg-resistant tumor cell lines using protein kinase C (PKC) agonists phorbol myristate acetate (PMA) or Bryostatin.

Results: Tumor cells were co-cultured with various concentrations of PMA and IFNg for 48 hr. The expression of MHCII antigens and receptors IFNgR1 and IFNgR2 was determined by flow cytometry. We showed that the presence of as little as 0.1 ng/ml of PMA in tissue culture restored the ability of weakly inducible LS1034 colon carcinoma cells to express MHCII in response to IFNg (100 - 10,000 IU/ml) in a dose-dependent manner. Likewise, Bryostatin 1, as low as 10 ng/ml produced a 5-6 fold upregulation of MHCII. The effect of PMA was not observed in two other poorly responding cell lines, MSTO-211H mesothelioma and HepG2 hepatocellular carcinoma, and was abrogated by relatively high concentrations of PKC inhibitors staurosporine (100 nM) and GF 109203X (1,000 nM). Both surface and intracellular staining of all cell lines with antibodies against IFNgR1 and IFNgR2 failed to detect any increase in IFNg receptor expression following incubation with PMA.

Conclusion: In this study we showed that IFNg-inducibility of MHCII antigens in weakly inducible LS1034 colorectal carcinoma cell line can be rescued by concomitant incubation with PKC agonists. Bryostatin 1 may be considered for further investigation of IFNg-dependent MHCII induction in resistant tumors in vivo.

No MeSH data available.


Related in: MedlinePlus