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Autocrine stimulation of TNF-alpha mRNA expression in HL-60 cells
Abstract:
We have employed a human promyelocytic leukemic cell line, HL-60, to investigate the conditions that regulate TNF-alpha gene expression in vitro. Using a cloned TNF-alpha specific cDNA probe, we show by Northern blot analysis that TNF-alpha mRNA rapidly accumulates in HL-60 cells following treatment with phorbol myristate acetate (PMA). TNF-alpha mRNA levels peak at 1-2 hours and then decline to base-line levels within 8 hours. TNF-alpha protein levels as detected in the supernatants of PMA-stimulated HL-60 cells peak at 2 hours and decline within 24 hours. Cytokines of recombinant source like interferon-alpha and -gamma, and, more intruigingly, TNF-beta as well as TNF-alpha itself are also able to induce transient TNF-alpha mRNA expression in HL-60 cells. In the presence of a protein kinase inhibitor, neither PMA nor any of the cytokines used are able to induce TNF-alpha mRNA accumulation, indicating that protein kinases may be crucially involved in signal transduction leading to activation of the TNF-alpha gene. The data presented provide new insights into the control of TNF-alpha gene expression suggesting regulatory functions of T- and B-cell derived lymphokines as well as a TNF-alpha-mediated positive feedback mechanism.
Insights
Tumor necrosis factor-alpha (TNF-alpha) gene expression in HL-60 cells is rapidly induced by phorbol myristate acetate (PMA) and certain cytokines. Protein kinases are crucial for this TNF-alpha gene activation.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Tumor necrosis factor-alpha (TNF-alpha) is a key cytokine involved in inflammation and immune responses.
- Understanding the regulation of TNF-alpha gene expression is critical for developing targeted therapies.
Purpose of the Study:
- To investigate the in vitro conditions regulating TNF-alpha gene expression in human promyelocytic leukemic (HL-60) cells.
- To elucidate the signaling pathways involved in TNF-alpha gene activation.
Main Methods:
- Northern blot analysis was used to detect TNF-alpha mRNA accumulation.
- HL-60 cells were treated with phorbol myristate acetate (PMA) and various recombinant cytokines.
- Protein kinase inhibitors were employed to assess the role of kinases in signal transduction.
Main Results:
- PMA treatment rapidly induced TNF-alpha mRNA and protein expression in HL-60 cells, with peak mRNA at 1-2 hours and protein at 2 hours.
- Recombinant interferon-alpha, interferon-gamma, TNF-beta, and TNF-alpha also induced transient TNF-alpha mRNA expression.
- Inhibition of protein kinases blocked PMA- and cytokine-induced TNF-alpha mRNA accumulation.
Conclusions:
- TNF-alpha gene expression is tightly regulated in HL-60 cells.
- Protein kinases play a critical role in the signal transduction pathway leading to TNF-alpha gene activation.
- Lymphokines and TNF-alpha itself may exert regulatory functions on TNF-alpha gene expression, suggesting potential feedback mechanisms.