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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

Experimental conditions for measuring MHCII-specific cellular fluorescence by flow cytometry. Tumor cells were removed from plastic with 0.25% trypsin and 0.53 mM EDTA, washed, stained with FITC-conjugated monoclonal antibodies (mAb) and analysed on a FACSCalibur™ flow cytometer. Regions R1, R2 and R3 were drawn to exclude debris (G), dead cells (A, B, C) and cellular aggregates (D, E, F). Panels A and D show cells stained with propidium iodide alone. Panels B and E show cells stained with propidium iodide and the isotype-matched control mAb (IgG2a-FITC, 1.0 μg/50 μl). Panel C and F show cells stained with propidium iodide and the mAb against human HLA-DR,DP,DQ (anti-MHCII-FITC, clone Tü39, 0.25 μg/50 μl). Panel H shows frequency distributions of cells that passed R1 & R2 & R3 logical gate. M1, M2 and M3 are the median values of autofluorescence peak (M1 = 222), isotype control peak (M2 = 234) and HLA-DR peak (M3 = 445).
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Figure 8: Experimental conditions for measuring MHCII-specific cellular fluorescence by flow cytometry. Tumor cells were removed from plastic with 0.25% trypsin and 0.53 mM EDTA, washed, stained with FITC-conjugated monoclonal antibodies (mAb) and analysed on a FACSCalibur™ flow cytometer. Regions R1, R2 and R3 were drawn to exclude debris (G), dead cells (A, B, C) and cellular aggregates (D, E, F). Panels A and D show cells stained with propidium iodide alone. Panels B and E show cells stained with propidium iodide and the isotype-matched control mAb (IgG2a-FITC, 1.0 μg/50 μl). Panel C and F show cells stained with propidium iodide and the mAb against human HLA-DR,DP,DQ (anti-MHCII-FITC, clone Tü39, 0.25 μg/50 μl). Panel H shows frequency distributions of cells that passed R1 & R2 & R3 logical gate. M1, M2 and M3 are the median values of autofluorescence peak (M1 = 222), isotype control peak (M2 = 234) and HLA-DR peak (M3 = 445).

Mentions: Cellular monolayers were rinsed 3 times with Ca/Mg-free PBS and incubated for 20 min at 37°C in Hanks' balanced salt solution containing 0.25% trypsin, 1 mM EDTA and 25 mM HEPES. Detached cells were washed twice with staining buffer (PBS containing 10% FCS, 0.1% sodium azide and 25 mM HEPES, pH 7.4) and stained as described previously [49]. Briefly, cells were transferred into the wells of round-bottom 96-well plates, the plates were centrifuged at 200 g for 30 seconds, the supernatant removed by shaking, and the cell pellets resuspended in 50 μl of staining buffer containing saturating concentration of anti-MHCII-FITC. After 30-min incubation at 4°C, cells were washed twice, resuspended in staining buffer and kept on ice before analysis on a flow cytometer (FACSCalibur™, Becton Dickinson Immunocytometry Systems). Immediately before analysis, 1 μg/ml propidium iodide was added to exclude dead cells. Matching isotype control mAb (IgG2a-FITC) was used at the same (0.25 μg/well) or a higher (1.0 μg/well) concentration as the specific antibody. The lack of staining of controls demonstrated that non-specific binding of IgG2a-FITC to PI-negative cells was negligible in all experimental groups (Fig 8B,8E,8H). Staining for cell surface IFNg receptors was performed similarly, except that cells were first incubated with biotin-conjugated mAb's (specific or isotype-matched) for 30 min, washed 2 times and then stained with SA-Alf488. Staining of fresh and trypsin-treated monocytes demonstrated that epitopes recognized by the mAb's against MHCII, IFNgR1 and IFNgR2 were resistant to 30 min digestion with 0.25% trypsin.


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)

Experimental conditions for measuring MHCII-specific cellular fluorescence by flow cytometry. Tumor cells were removed from plastic with 0.25% trypsin and 0.53 mM EDTA, washed, stained with FITC-conjugated monoclonal antibodies (mAb) and analysed on a FACSCalibur™ flow cytometer. Regions R1, R2 and R3 were drawn to exclude debris (G), dead cells (A, B, C) and cellular aggregates (D, E, F). Panels A and D show cells stained with propidium iodide alone. Panels B and E show cells stained with propidium iodide and the isotype-matched control mAb (IgG2a-FITC, 1.0 μg/50 μl). Panel C and F show cells stained with propidium iodide and the mAb against human HLA-DR,DP,DQ (anti-MHCII-FITC, clone Tü39, 0.25 μg/50 μl). Panel H shows frequency distributions of cells that passed R1 & R2 & R3 logical gate. M1, M2 and M3 are the median values of autofluorescence peak (M1 = 222), isotype control peak (M2 = 234) and HLA-DR peak (M3 = 445).
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Related In: Results  -  Collection

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Figure 8: Experimental conditions for measuring MHCII-specific cellular fluorescence by flow cytometry. Tumor cells were removed from plastic with 0.25% trypsin and 0.53 mM EDTA, washed, stained with FITC-conjugated monoclonal antibodies (mAb) and analysed on a FACSCalibur™ flow cytometer. Regions R1, R2 and R3 were drawn to exclude debris (G), dead cells (A, B, C) and cellular aggregates (D, E, F). Panels A and D show cells stained with propidium iodide alone. Panels B and E show cells stained with propidium iodide and the isotype-matched control mAb (IgG2a-FITC, 1.0 μg/50 μl). Panel C and F show cells stained with propidium iodide and the mAb against human HLA-DR,DP,DQ (anti-MHCII-FITC, clone Tü39, 0.25 μg/50 μl). Panel H shows frequency distributions of cells that passed R1 & R2 & R3 logical gate. M1, M2 and M3 are the median values of autofluorescence peak (M1 = 222), isotype control peak (M2 = 234) and HLA-DR peak (M3 = 445).
Mentions: Cellular monolayers were rinsed 3 times with Ca/Mg-free PBS and incubated for 20 min at 37°C in Hanks' balanced salt solution containing 0.25% trypsin, 1 mM EDTA and 25 mM HEPES. Detached cells were washed twice with staining buffer (PBS containing 10% FCS, 0.1% sodium azide and 25 mM HEPES, pH 7.4) and stained as described previously [49]. Briefly, cells were transferred into the wells of round-bottom 96-well plates, the plates were centrifuged at 200 g for 30 seconds, the supernatant removed by shaking, and the cell pellets resuspended in 50 μl of staining buffer containing saturating concentration of anti-MHCII-FITC. After 30-min incubation at 4°C, cells were washed twice, resuspended in staining buffer and kept on ice before analysis on a flow cytometer (FACSCalibur™, Becton Dickinson Immunocytometry Systems). Immediately before analysis, 1 μg/ml propidium iodide was added to exclude dead cells. Matching isotype control mAb (IgG2a-FITC) was used at the same (0.25 μg/well) or a higher (1.0 μg/well) concentration as the specific antibody. The lack of staining of controls demonstrated that non-specific binding of IgG2a-FITC to PI-negative cells was negligible in all experimental groups (Fig 8B,8E,8H). Staining for cell surface IFNg receptors was performed similarly, except that cells were first incubated with biotin-conjugated mAb's (specific or isotype-matched) for 30 min, washed 2 times and then stained with SA-Alf488. Staining of fresh and trypsin-treated monocytes demonstrated that epitopes recognized by the mAb's against MHCII, IFNgR1 and IFNgR2 were resistant to 30 min digestion with 0.25% trypsin.

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