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Cytostasis is required for IL-1 induced nitric oxide production in transformed hamster fibroblasts
N Lavnikova1, A Lakhotia, N Patel
1Rutgers University, Piscataway, New Jersey 08855-0789, USA.
Insights
Interleukin-1 (IL-1) inhibits tumor cell growth, but nitric oxide is not the primary mediator. IL-1-induced cytostasis occurs independently of nitric oxide production in STHE cells.
Area of Science:
- Cell Biology
- Immunology
- Cancer Research
Background:
- Interleukin-1 (IL-1) is a proinflammatory cytokine with known effects on tumor cell proliferation.
- IL-1 can induce nitric oxide (NO) production in various cell types.
- The role of NO in IL-1-mediated growth inhibition of tumor cells remains unclear.
Purpose of the Study:
- To investigate the involvement of nitric oxide (NO) in Interleukin-1 (IL-1) induced growth inhibition of spontaneously transformed hamster embryonic fibroblasts (STHE cells).
Main Methods:
- STHE cells were treated with IL-1 alpha and IL-1 beta.
- Nitric oxide production was measured.
- Cell proliferation was assessed using 3H-thymidine incorporation.
- Nitric oxide synthase inhibitor NG-monomethyl-1-arginine (L-NMMA) and neutralizing antibodies to IL-1 and TNF-alpha were used.
Main Results:
- IL-1 stimulated NO production and reduced proliferation in high-density STHE cell cultures.
- Cytostasis occurred before significant NO accumulation, and low IL-1 doses induced cytostasis without NO synthesis.
- IL-1 inhibited DNA synthesis in low-density cultures without NO production.
- L-NMMA partially reduced IL-1-induced cytostasis in high-density cultures.
- Anti-IL-1 antibodies blocked IL-1 effects; anti-TNF-alpha partially suppressed IL-1 activity.
- Non-proliferating cells produced more NO than exponentially growing cells.
Conclusions:
- Nitric oxide is not a major mediator of IL-1 induced cytostasis in STHE cells.
- Cytostasis appears to be a prerequisite for NO synthesis in these cells.
- Endogenous TNF-alpha may play a role in IL-1 induced cytostasis and NO production.
Abstract:
Interleukin-1 (IL-1) is known to inhibit proliferation in some tumor cells. This proinflammatory cytokine also induces nitric oxide production in a variety of cell types. In the present studies we determined if nitric oxide is involved in IL-1 induced growth inhibition in spontaneously transformed hamster embryonic fibroblasts (STHE cells). Both IL-1 alpha and IL-1 beta were found to stimulate nitric oxide production and to reduce 3H-thymidine (TdR) incorporation in high density cultures of STHE cells. However, maximal cytostasis was observed at least 24 h before significant amounts of nitric oxide accumulated in the cultures. In addition, doses of IL-1 which were too low to stimulate nitric oxide synthesis were effective in inducing cytostasis. Furthermore, in low density cultures of STHE cells, IL-1 inhibited DNA synthesis without inducing nitric oxide production. The nitric oxide synthase inhibitor NG-monomethyl-1-arginine (L-NMMA) had no effect on proliferation of cells plated at low density. In contrast, L-NMMA treatment resulted in a 40-60% reduction in IL-1 induced cytostasis in high density cultures. Neutralizing antibodies to IL-1 were found to completely block IL-1 induced cytostasis and nitric oxide production in cells plated at both densities. Although anti-IL-1 alpha and anti-IL-1 beta antibodies were highly specific and did not cross react, anti-tumor necrosis factor-alpha (TNF-alpha) antibody was able to partially suppress activation of STHE cells by both IL-1 alpha and IL-1 beta. These data suggest a potential involvement of endogenous TNF-alpha in IL-1 induced cytostasis and nitric oxide production. Exponentially growing STHE cells produced six-times less nitric oxide than non-proliferating cells. A ten-fold excess of 1-arginine was found to stimulate nitric oxide synthesis, an action that was independent of the rate of cellular proliferation. Taken together these data suggest that nitric oxide is not a major mediator of IL-1 induced cytostasis in STHE cells. Moreover, cytostasis appears to be required for nitric oxide synthesis in these cells.