Hypoxic inducible factor 1alpha, extracellular signal-regulated kinase, and p53 are regulated by distinct threshold concentrations of nitric oxide - PubMed (original) (raw)

Comparative Study

. 2004 Jun 15;101(24):8894-9.

doi: 10.1073/pnas.0400453101. Epub 2004 Jun 3.

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Comparative Study

Hypoxic inducible factor 1alpha, extracellular signal-regulated kinase, and p53 are regulated by distinct threshold concentrations of nitric oxide

Douglas D Thomas et al. Proc Natl Acad Sci U S A. 2004.

Abstract

NO produced in tumors can either positively or negatively regulate growth. To examine this dichotomy, effects of NO concentration and duration on the posttranslational regulation of several key proteins were examined in human breast MCF7 cells under aerobic conditions. We found that different concentration thresholds of NO appear to elicit a discrete set of signal transduction pathways. At low steady-state concentrations of NO (<50 nM), extracellular signal-regulated kinase (ERK) phosphorylation was induced via a guanylate cyclase-dependent mechanism. Hypoxic inducible factor 1alpha (HIF-1alpha) accumulation was associated with an intermediate amount of NO (>100 nM), whereas p53 serine 15 phosphorylation occurred at considerably higher levels (>300 nM). ERK phosphorylation was transient during NO exposure. HIF-1alpha stabilization paralleled the presence of NO, whereas p53 serine 15 phosphorylation was detected during, and persisted after, NO exposure. The dose-dependent effects of synthetic NO donors were mimicked by activated macrophages cocultured with MCF7 cells at varying ratios. ERK and HIF-1alpha activation was similar in breast cancer cell lines either mutant (MB231) or null (MB157) in p53. The stabilization of HIF-1alpha by NO was not observed with increased MCF7 cell density, demonstrating the interrelationship between NO and O(2) consumption. The findings show that concentration and duration of NO exposure are critical determinants in the regulation of tumor-related proteins.

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Figures

Fig. 1.

Fig. 1.

Temporal relationship between NO exposure and protein accumulation in MCF7 cells. Representative immunoblot of p53 P-Ser-15, HIF-1α, and pERK from MCF7 cell protein extracts after NO exposure (n > 3). Cells grown to 85% confluence in 150-mm Petri dishes were serum-starved overnight, treated with Sper/NO (100 μM), and harvested at the indicated time points. D, decomposed Sper/NO, 100 μM for 12 h; representative of all time points (data not shown).

Fig. 2.

Fig. 2.

Protein accumulation as a function of NO concentration in MCF7 cells. (A) Cells grown to 85% confluence in 150-mm Petri dishes were serum-starved overnight and treated with Sper/NO as indicated. Time points were chosen corresponding to maximal protein accumulation (Fig. 1). D, decomposed Sper/NO (100 μM). (B) Cells were grown and treated with Sper/NO as in A. NO concentrations were determined from 100-μl sample aliquots of medium withdrawn from the Petri dish by gas-tight syringe without agitation and analyzed by chemiluminescence at the indicated time points. Representative data are shown as the mean ± SE (n = 3).

Fig. 3.

Fig. 3.

Comparison of HIF-1α and p53 P-Ser-15 accumulation by NO from either Sper/NO or activated macrophages. MCF7 cells were grown as in Fig. 1. (A) Four-hour treatment of MCF7 cells with either Sper/NO (50 and 100 μM) or ratios of activated NO-producing ANA-1 macrophages (MCF7:ANA-1) as indicated. 1:8 + AG = MCF7:ANA-1 1:8 + iNOS inhibitor aminoguanidine. (B) NO concentration from cocultured MCF7 and ANA-1 cells was determined as described in Fig. 2_B_. Representative data are shown (n = 3).

Fig. 4.

Fig. 4.

HIF-1α and p53 P-Ser-15 accumulation in response to various durations of NO exposure and real-time quantification of NO concentration. MCF7 cells were grown as in Fig. 1 and exposed to the NO donors. (A) 100 μM DEA/NO. (B) 100 μM Sper/NO. (C) 1,000 μM DETA/NO. NO steady-state levels were quantified as in Fig. 2_B_.

Fig. 5.

Fig. 5.

cGMP accumulation in MCF7 cells in response to various concentrations of the NO donor Sper/NO. MCF7 cells were grown to 50% confluency in a 96-well microtiter plates, serum-starved overnight, and treated with Sper/NO for the indicated time points (n = 3).

Fig. 6.

Fig. 6.

Effect of Hsp90 inhibition on NO-induced protein stabilization. MCF7 cells were grown as in Fig. 1. Cells were treated with Sper/NO (100 μM) ± geldanamycin (10 μM) and harvested at the indicated time points. HIF-1α and p53 P-Ser-15 were undetectable in untreated controls (data not shown).

Fig. 7.

Fig. 7.

Effect of p53 status on NO-mediated HIF-1α stabilization. MB 231 and MB 157 breast cancer cells were grown as in Fig. 1, treated with Sper/NO (100 μM), and harvested at the indicated time points. MB 157 cells were grown in hypoxia for 4 h without Sper/NO as a HIF-1α positive control (4 hypoxia). A p53 P-Ser-15-positive MCF7 sample was blotted for an internal p53 control [p53 (+) control].

Fig. 8.

Fig. 8.

Electrochemical detection of NO in the presence of MCF7 cells. MCF7 cells were added in suspension (3 × 106/ml) as described in Materials and Methods. O2 was monitored continuously for 120 min (data not shown). Cells were isolated, and proteins were immunoblotted for HIF-1α. (A) Normoxia (≈21% O2); (B) hypoxia (<1% O2); (C) intermediate (≈10% O2). Representative NO electrode data for condition C are shown (n = 3). Sper/NO (100 μM) was added to the chamber (1). MCF7 cells were added after a steady-state NO level was achieved (2).

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References

    1. Juang, S. H., Xie, K., Xu, L., Shi, Q., Wang, Y., Yoneda, J. & Fidler, I. J. (1998) Hum. Gene. Ther. 9, 845–854. - PubMed
    1. Reveneau, S., Arnould, L., Jolimoy, G., Hilpert, S., Lejeune, P., Saint-Giorgio, V., Belichard, C. & Jeannin, J. F. (1999) Lab. Invest. 79, 1215–1225. - PubMed
    1. Pervin, S., Singh, R. & Chaudhuri, G. (2001) Proc. Natl. Acad. Sci. USA 98, 3583–3588. - PMC - PubMed
    1. Scott, D. J., Hull, M. A., Cartwright, E. J., Lam, W. K., Tisbury, A., Poulsom, R., Markham, A. F., Bonifer, C. & Coletta, P. L. (2001) Gastroenterology 121, 889–899. - PubMed
    1. Wei, D., Richardson, E. L., Zhu, K., Wang, L., Le, X., He, Y., Huang, S. & Xie, K. (2003) Cancer Res. 63, 3855–3859. - PubMed

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