Tara Leigh Nelson and Dr. Allan M. Judd, Zoology
Cytokines are extracellular signaling proteins or peptides that act as local mediators in cell-cell communication. They regulate several endocrine systems and function in the immune process. These agents mediate cellular proliferation within endocrine tissues, regulate hormone secretion, and control aspects of inflammation (1). Interleukin-1a (IL- 1a), IL-6 and tumor necrosis factor-a (TNF-a) are present in normal adrenal glands of rats, bovine and humans. They stimulate and inhibit adrenal hormones. Growth and differentiation of the adrenal gland may also be affected by cytokines. The study of the effects of these cytokines is important because adrenal tumors may cause hypersecretion of the adrenal cortex leading to such disorders as Cushing’s disease (hyperadrenalism).
My desired goal was to ascertain the effects of increased levels of IL-1a, IL-6 and TNF- a on the growth of the human H295R adrenal carcinoma cell line (2). I evaluated this by determining the result each cytokine has on cell number.
H295R cells were cultured into four rows of wells on three 96-well plates containing 0.2 ml of H295R assay medium and one of the following: assay medium (control), Interleukin-1a (10 hg/ml), Interleukin-6 (10 rg/ml) or tumor necrosis factor-a (1 mg/ml). An additional row was used containing only assay medium to serve as a blank for the experiment. Ten thousand cells/well were left to culture for 72 hours during the first trial. In the second trial I used 20,000 cells/well and left them to culture for 48 hours. During the third trial 20,000 cells/well were left to culture for 72 hours.
One hundred ml of culture medium was removed from the top of each well after each of the trials respective culture time. Ten ml (5 mg/ml) of 3-(4,5-dimethyl thiazol-2-gl)-2,5- diphenyl tetrasodium bromide (MTT) was added to each well for four hours. Next, 100 ml of isopropanol with 0.04N HCL was added to each well. Active mitochondria cleaved the yellow MTT and formed a dark blue formazon product that is soluble in isopropanol. The viable cells were measured using the production of blue color as an index. An ELISA plate reader determined the absorbance in each well at 570 hm. The absorbance of a standard curve of 1,000 to 100,000 cells per well was used as a basis of comparison to determine mean cell number and response in cell growth.
All three cytokines appeared to cause a decrease in cell number as determined by absorbance using the ELISA plate reader. TNF-a had the most negative effect on cell growth, followed by IL-6. IL-1a decreased cell number the least, and even caused a slight increase in cell growth during Trial 2 (20,000 cells/48 hrs.).
The following table indicates the findings of my experiment
My theory is that TNF-a is a growth inhibitor of the human H295R adrenal tumor cell line. Perhaps this is because it inhibits insulin-like growth factor II and H19 gene expression, as it has been shown to do in human fetal adrenal cell cultures (3). IL-6 has also shown to decrease cell number. This is contrary to my initial hypothesis that IL-6 would increase the rate of growth of H295R cells due to the reduced expression of H19, a putative tumor suppressor gene. The results of IL-1a’s effects on H295R are inconclusive. Because the hormone caused an increase in cell number in trial 2, I cannot conclude whether IL-1a is indeed a cell growth suppressor as suggested by trials 1 and 3.
Further studies on this subject are needed. Various concentrations of each cytokine should be used to determine whether different concentrations cause a different response in cell growth. Variable culture times and various cell concentrations should also be tested. Re-testing these variables would lead to more conclusive results. Future research could lead to the prevention and treatment of adrenal tumors that cause hypersecretion of the adrenal cortex.
References
- Spangelo BL, Judd AM, Call GB, Zumwalt J, Gorospe WC: Role of the Cytokines in the Hypothalamic-Pituitary-Adrenal and Gonadal Axes. Neuroimmunomodulation 1995;2:299-312.
- Rainey WE, Bird IM, Mason JI: The NCI-H295R cell line: a pluripotent model for human adrenocortical studies. Mol and Cell Endo 1990;100:45-50.
- Ilvesmäki V, Jäätelä M, Saksela E, Voutilainen R: Tumor necrosis factor-a and interferon-g inhibit insulin-like growth factor II gene expression in human fetal adrenal cell cultures. Mol Cell Endocrinal 1993;91:59-65.