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MEK and ERK inhibitors enhance the anti-proliferative effect of interferon-2b¹

Institute of Human Virology, University of Maryland Biotechnology Institute, Baltimore, Maryland, USA

²Correspondence: Institute of Human Virology, University of Maryland Biotechnology Institute, 725 West Lombard St., Baltimore, MD 21201, USA. E-mail: zella{at}umbi.umd.edu

SPECIFIC AIMS

Interferon (IFN-) is used in clinical practice for the treatment of several tumors, including hematopoietic malignancies. The aim of this study was to demonstrate that short-term exposure to IFN- reduces function of the MEK/ERK pathway (important for T cell proliferation) and that molecules targeting such pathway increase the anti-proliferative effect of IFN- in vitro. Our data may eventually lead to new clinical strategies to strengthen its anti-cancer effect.

PRINCIPAL FINDINGS

1. IFN-2b inhibits proliferation of human primary CD4+ T cells IFN-2b by preventing exit from G0/G1 and entry into S phase of the cell cycle
Primary resting CD4+ T cells stimulated with anti-CD3 plus IL-2 in the presence or absence of IFN- were collected every 24 h over a 5 day period. Between 24 and 48 h of stimulation, the number of cells in G0/G1 in the control cultures dropped rapidly from 85% to 62%, reaching a minimum of 58% at 72 h. At the same time there was a 10-fold increase (3–30%) in the number of cells in S phase in the untreated cultures. This indicates that the cells were efficiently exiting from G0 and progressing through G1 and into S phase. On the contrary, on treatment with IFN-, the number of cells in G0/G1 decreased very slowly from 85% at 24 h to 73% at 96 h poststimulation. The number of cells entering the S phase was strongly reduced and was never more than 20%. These results indicate that IFN- hinders the transition from G0/G1 to S phase in CD4+ T cells, with an overall 40% decrease in the total number of cells able to perform this task over the 5 day period of culture.

2. Short-treatment with IFN-2b produces long-term effects on cell cycle progression
An initial delay of 2–6 h in the addition of IFN- after stimulation with anti-CD3 and IL-2 decreased the inhibitory effect of IFN- by 30%, as assessed by the number of cells in G0/G1 at day 3 compared to the negative and positive controls. A delay of 12 h decreased the inhibitory effect of IFN- by 50%. A retard in the onset of the treatment with IFN- also reduced its anti-proliferative effect. A brief treatment with IFN- during the first 2–6 h of stimulation with anti-CD3 plus IL-2 was sufficient to inhibit the exit from G0/G1 as much as in the positive control. This effect was not significantly increased when the cells were treated with IFN- for longer periods or if they were primed with IFN- for different lengths of time (2–24 h) before stimulation with anti-CD3 and IL-2. The proliferative ability of cells treated with IFN- during the first 2–6 h of stimulation with anti-CD3 plus IL-2 was strongly impaired, just as in the positive controls. These results indicate that the anti-proliferative signal of IFN- is fully delivered early and within a very narrow window of time after stimulation with anti-CD3 and IL-2. Removal of IFN- after this brief initial window still results in a strong impairment in cell cycle progression.

3. Short-term treatment with IFN-2b inhibits activation of the MEK/ERK pathway without affecting Raf-1 and Ras activity
We determined the minimal time required for IFN- treatment to exert its long-term effects on ERK1/2 and MEK1 activity. CD4+ T cells were stimulated for 2, 6, or 12 h with anti-CD3 either alone or with IFN-. Exposure to IFN- as brief as 2–6 h was sufficient to decrease substantially the phosphorylation of ERK1/2 (Fig. 1 A) and MEK (Fig. 1B ) whereas total levels of these proteins remained unchanged. Lower panels of Fig. 1A, B report average and SD (in relative units) of phospho-ERK1/2 and phospho-MEK1 from four independent experiments. These results suggest that IFN- sets in motion a cascade of events within the first 2–6 h of treatment leading to impairment of MEK/ERK function. These events eventually lead to impaired cell proliferation observed 24–48 h later even after early removal of IFN-.

View larger version (19K): [in this window] [in a new window]   Figure 1. Relationship between duration of treatment with IFN-2b and MEK/ERK phosphorylation in purified CD4+ T cells. CD4+ T cells were purified by negative selection from total PBMC of healthy seronegative donors. 4 x 107 cells were stimulated with 250 ng/ml anti-CD3 alone or in the presence of 10 ng/ml IFN-2b. At 2, 6, and 12 h, whole-cell lysates were prepared and the amounts of phosphorylated of ERK1/2 (A) and MEK1 (B) were assessed by Western blot. Results are representative of experiments carried out with cells from four donors. X-ray films obtained in the four independent experiments were subjected to densitometric analysis and results are expressed as arbitrary relative units in the lower panels. Mean and SD are shown.

Treatment with IFN- did not affect Raf-1 kinase activity in our in vitro assay system. We also measured GTPase activity of Ras in anti-CD3 x CD4-stimulated cells treated with IFN- compared with control samples. Once again, we failed to observe any significant and consistent difference in the levels of GTP- vs. GDP-bound Ras in the treated cells and control samples (not shown). These results indicate that short-term treatment with IFN- inhibits long-term MEK/ERK function without affecting Ras and Raf-1 activity.

4. IFN- and MEK/ERK inhibitors cooperatively reduce CD4+ T cell proliferation
CD4+ T cells were activated with anti-CD3 plus IL-2 and serial dilutions of IFN- were used in combination with inhibitors of MEK (PD98059) and ERK (hypericin). After 3 days, viable cells were counted by Trypan blue exclusion. A dose-dependent effect on cell proliferation was observed for each one of the compounds tested, with reduction ranging from 15–35%, with respect to the untreated control (Fig. 2 A). When low doses of IFN- (0.1–1.0 ng/ml) were combined with increasing amounts of PD98059, the anti-proliferative effect of IFN- was increased from 30–35% to 35–50% (P<0.05) (Fig. 2B, C ). However, with higher doses of IFN- (10 ng/ml) this additive effect progressively disappeared (Fig. 2C, D ). When IFN- and hypericin were combined, the additive effect was observed with all concentrations of IFN- used (P<0.05) (compare Fig. 2B, C, and D ). The anti-proliferative effect of IFN- was increased from 35–50% to 45–60% by the addition of hypericin. These results demonstrate that:1) the MEK/ERK pathway is a critical pathway target by IFN- to exert its anti-proliferative effect, and 2) combined treatment with compounds inhibiting MEK and ERK increases the anti-proliferative effect of IFN-.

View larger version (56K): [in this window] [in a new window]   Figure 2. IFN- cooperates with inhibitors of MEK and ERK to reduce CD4+ T cell proliferation. Purified CD4+ T cells were activated with anti-CD3 plus IL-2. At the time of stimulation, IFN- and inhibitors of MEK (PD98059) and ERK (hypericin), alone or in combination, were added to the culture medium. After 3 days, viable cells were counted by Trypan blue exclusion. A) Inhibition of cell proliferation after treatment with different concentration of IFN- (0.1, 1, and 10 ng/ml), PD98059 (0.1, 1, 10 µM), and hypericin (0.1, 1, and 10 nM). B–D) IFN- (0.1, 1, and 10 ng/ml, respectively) was used in combination with the indicated concentration of PD98059 and hypericin.

CONCLUSIONS

In a recent study we showed that IFN- inhibits cell proliferation as well as IL-2 production and IL-2 receptor function in CD4+ T cell costimulated with immobilized anti-CD3 plus anti-CD28 antibodies. In the present report we have extended our previous observations in the context of CD4+ T cells stimulated with anti-CD3 plus IL-2, confirming the anti-proliferative activity of IFN-. This indicates that the molecular mechanism(s) underlying this phenomenon affects a signaling pathway triggered in both stimulation conditions.

Our data presented here on the kinetics of cell cycle progression in cultures of CD4+ T cells treated with IFN- have revealed a marked reduction in the ability to exit G0/G1 and progress through S phase over a 5 day culture period. Similar to the effect on MEK/ERK activation, the inhibitory signal of IFN- on cell cycle dynamics does not require a treatment for extended periods of time, but must be supplied very early on stimulation through the TCR. A delay of only a few hours in treating cells with IFN- after the beginning of the TCR stimulation vanished or diminished the anti-proliferative effects of IFN-.

Our results suggest that IFN- can interfere with de novo protein synthesis and/or regulation of a specific gene(s) implicated in the regulation of MEK phosphorylation (Fig. 3 ). This hypothesis may explain our observation that treatment with IFN- during the first few hours of stimulation with anti-CD3 plus IL-2 and subsequent removal of the treatment result in a long-lasting inhibitory effect on cell proliferation and MEK/ERK function. Such brief treatment may be sufficient to induce or prevent the expression of a specific gene(s), affecting MEK/ERK activation and ultimately resulting in the anti-proliferative effect of IFN-. This hypothesis would also explain the observation that inhibition of MEK/ERK function does not seem to involve an impairment in function of the upstream regulators of MEK, ERK, Ras, and Raf-1. Having identified a phosphorylative cascade implicated in cellular proliferation as a target for IFN-, we reasoned that inhibitors of this cascade could cooperate with IFN- to increase its anti-proliferative effect. Two inhibitors of MEK and ERK (PD98059 and hypericin) proved to be effective. PD98059 has been demonstrated to reduce the growth of a colon cancer cell line in an in vivo model. Hypericin is currently used to treat several types of cancers including lymphoma, basal and squamous cell carcinoma, and glioma. In our in vitro system, a significant effect on cell proliferation was observed when PD98059 was combined with low levels of IFN-. In contrast, when higher levels of IFN- were used, PD98059 did not exert an additive effect. Treatment with hypericin, on the other hand, significantly increased the anti-proliferative effect of IFN- in all concentrations tested. Another molecular target of hypericin is protein kinase C, which cooperates in MEK/ERK activation. This could partially explain the potent additive anti-proliferative effect we observed in vitro in combination with IFN-.

View larger version (14K): [in this window] [in a new window]   Figure 3. IFN- regulates MEK/ERK pathway activity and specific inhibitors of MEK and ERK increase the IFN- anti-proliferative effect. After binding to the specific receptor (1), IFN- could either interfere with de novo protein synthesis (2) and/or regulation of a specific gene(s) (3) implicated in the regulation of MEK phosphorylation (4). Molecules targeting the MEK/ERK pathway (5) can potentiate the anti-proliferative effect of IFN- (6).

Our data constitute proof of the concept that molecules targeting the MEK/ERK pathway can potentiate the anti-proliferative effect of IFN- (Fig. 3) . This could lead to new multidrug therapies aimed at strengthening the anti-tumor effect of IFN-. Further studies into the mechanism(s) responsible for the IFN-mediated effect on the MEK/ERK pathway may provide additional targets for cancer therapy.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0120fje; to cite this article, use FASEB J. (August 19, 2002) 10.1096/fj.02-0120fje

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