Figure 4: Cervical cancer cell lines express MICA, MICB and NKG2D. CALO and INBL cells (1 × 107) were lysed proteins immunoprecipitated and equal amounts of protein from total lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The blots were developed with either polyclonal anti-MIC antibodies (A) or monoclonal anti-NKG2D antibodies (B) and an appropriate secondary antibody conjugated to HRP for chemiluminescence detection. Flow cytometric analysis of NKG2D expression in cervical carcinoma cell lines after 72 h induction with 10 ng MICB (C). We used only MICB to induce the expression of NKG2D because we previously obtained that MICB was a better inducer of myelomonocytic cell proliferation than MICA. Graphs show NKG2D levels (solid line) and isotype controls (dotted line).

Mentions: In order to evaluate the capacity of other tumor cell types to express MICA and MICB, as well as NKG2D, we evaluated the possible expression of these proteins in two human epithelial cervical cancer cell lines, CALO and INBL, using polyclonal antibodies against MICA/MICB and anti-NKG2D for western blot and flow cytometric analyses. Our results show that MICA, MICB and NKG2D were expressed in both cell lines (Figs. 4A and 4B). It is interesting to mention that when flow cytometric analysis for NKG2D expression was performed after the cells were activated for 72 h by MICB, only a small minority of the cells exhibited high NKG2D expression, while the majority of the cells expressed low levels of the receptor (Figure 4C). The presence of NKG2D was further evaluated by immunohistochemical analysis, which revealed a reproducible pattern of staining in both cervical cancer cell lines (Figure 5). We also evaluated if CALO and INBL secreted MICA and MICB into their culture media. For this purpose, we seeded 5 × 103 cells for up to eight days and detected significant amounts of MICA and MICB in the CM by ELISA; the concentration of MICA AND MICB increased during the first five days in culture (Figure 6).

Weiss-Steider B, Soto-Cruz I, Martinez-Campos CA, Mendoza-Rincon JF - J. Exp. Clin. Cancer Res. (2011)

Bottom Line: Values were considered significantly different if p < 0.05.THP-1 and U-937 produce and secrete the stress MICA and MICB as shown by Western Blot of lysed cells and by ELISA of their conditioned media.By Western Blot and flow cytometry we found that these cells also express the receptor NKG2D.By Western Blot, flow cytometry and immunocytochemistry we also found that these cells express NKG2D.Our novel results that tumor cells can simultaneously secrete MIC molecules and express their receptor, and to be induced for proliferation by these stress molecules, and that tumor epithelial cells can also express the NKG2D receptor that was thought to be exclusive of NK and cytotoxic lymphocytes is discussed as a possible mechanism of immunological escape and of tumor growth induction.

Affiliation: Laboratorio de Oncología Molecular, Unidad de Diferenciación Celular y Cáncer, FES-Zaragoza, Universidad Nacional Autónoma de México, Ciudad de México 09230, Mexico.

Abstract: Cancer cells are known to secrete the stress molecules MICA and MICB that activate cytotoxicity by lymphocytes and NK cells through their NKG2D receptor as a mechanism of immunological defense. This work was undertaken to evaluate if cancer cells can also express this receptor as a possible mechanisms of depletion of MIC molecules and thus interfere with their immune recognition.Myelomonocytic leukemic (TPH-1 and U-937) and cervical cancer (CALO and INBL) cell lines were evaluated by Western Blot, ELISA, flow cytometry and immunocytochemistry to evaluate their capacity to express and secrete MICA and MICB and to be induced to proliferate by these molecules as well as to express their receptor NKG2D. Statistical analysis was performed by two-way ANOVA for time course analysis and Student's t-test for comparison between groups. Values were considered significantly different if p < 0.05.THP-1 and U-937 produce and secrete the stress MICA and MICB as shown by Western Blot of lysed cells and by ELISA of their conditioned media. By Western Blot and flow cytometry we found that these cells also express the receptor NKG2D. When THP-1 and U-937 were cultured with recombinant MICA and MICB they exhibited a dose dependent induction for their proliferation. CALO and INBL also produce MICA and MICB and were induced to proliferate by these stress molecules. By Western Blot, flow cytometry and immunocytochemistry we also found that these cells express NKG2D.Our novel results that tumor cells can simultaneously secrete MIC molecules and express their receptor, and to be induced for proliferation by these stress molecules, and that tumor epithelial cells can also express the NKG2D receptor that was thought to be exclusive of NK and cytotoxic lymphocytes is discussed as a possible mechanism of immunological escape and of tumor growth induction.