Carcinogenesis

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Carcinogenesis, Vol. 20, No. 9, 1869-1872, September 1999
© 1999 Oxford University Press

Short Communications

Induction of cyclin-dependent kinase inhibitors and G₁ prolongation by the chemopreventive agent N-acetylcysteine

Ming Liu¹, Norbert M. Wikonkal¹ and Douglas E. Brash^1,2,3

¹ Department of Therapeutic Radiology and
² Department of Genetics, Yale University School of Medicine, New Haven, CT 06520-8040, USA

	Abstract

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Cyclin-dependent kinase (cdk) inhibitors, such as p16^INK4a and p21^WAF1/CIP1, often inhibit G₁ cyclin kinases and result in G₁ arrest. It has been suggested that p21^WAF1/CIP1 may also play a role in other chemopreventive activities such as DNA repair, slowdown of DNA replication and induction of cellular differentiation. In this report we demonstrate that the antioxidant N-acetylcysteine (NAC), a well-known chemopreventive agent, induces p16^INK4a and p21^WAF1/CIP1 gene expression and prolongs cell-cycle transition through G₁ phase. A portion of the G₁ arrest by NAC is governed by p16^INK4a; it is independent of p53. NAC's usual mechanism of increasing intracellular glutathione level is not required for the G₁ arrest. An antioxidant whose action is limited to scavenging radicals, Trolox, does not induce G₁ arrest. Taken together, these results suggest a potential novel molecular basis for chemoprevention by NAC.

Abbreviations: BSO, buthionine sulfoximine; cdk, cyclin-dependent kinase; GSH, glutathione; NAC, N-acetylcysteine.

	Introduction

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N-acetylcysteine (NAC) is known as a chemopreventive agent with anti-mutagenic and anti-tumorigenic activities in a variety of animal organs, including skin, lung, liver and colon (1–6). As an antioxidant, NAC can act as a scavenger of reactive oxidative intermediates either by itself or indirectly. In the latter role, it serves as a precursor for glutathione (GSH; -glutamylcysteinylglycine), the major intracellular thiol and antioxidant (7–9). NAC has been shown to block both DNA strand breakage and mutagenesis by DNA-damaging agents (10–12). NAC is a small molecule readily taken up by cells and is used in clinical treatments for chronic bronchitis and paracetamol poisoning (13).

In a previous study, we reported that NAC induces p53-dependent apoptosis selectively in oncogenically-transformed cells (14). In contrast, chain-breaking antioxidants such as vitamin E and Trolox lacked this activity. An increased GSH level was not required for apoptosis by NAC. Agents that induce apoptosis often cause cell-cycle arrest as well, typically at lower doses. Here, we investigate the effect of NAC on cell cycle-progression and on expression of two cyclin-dependent kinase (cdk) inhibitors. We find that NAC elicits p16^INK4a-mediated G₁-phase prolongation and upregulates p21^WAF1/CIP1 expression through a p53-independent pathway. These findings may have implications for the chemopreventive activities of NAC.

	Materials and methods

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Cell culture
The 308 papilloma cell line (wild-type p53) (15), the p53-null mouse keratinocyte cell line NHK-4 (16), and the mouse embryo fibroblast cell line (10)1 (17) were kindly provided by Drs S.Yuspa, W.Weinberg and A.Levine (National Cancer Institute, Rockefeller University), respectively. Human primary foreskin fibroblasts were supplied by the core facility of the Department of Dermatology, Yale School of Medicine, New Haven, CT. BALB/c 3T3 A31 cells were obtained from the American Type Culture Collection (Mannassas, VA). 308 papilloma cells were maintained in 0.05 mM Ca²⁺ Eagle's minimum essential medium with 10% fetal bovine serum, and all other cells were grown in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum. Primary keratinocytes from newborn mouse skin were prepared as described previously (18). p21^WAF1/CIP1 and INK4a knockout mice (19,20) were generously provided by Drs P.Leder (Harvard University, Boston, MA) and M.Serrano (Centro Nazional de Biotecnologia, Madrid), respectively.

Chemicals and cell treatments
NAC and Trolox were purchased from Sigma (St Louis, MO) and Aldrich (Milwaukee, WI), respectively. These were freshly dissolved in culture medium and adjusted to neutral pH if necessary. Culture confluence was maintained below 80%.

Northern and western blot analysis
Northern and western blot analyses were performed as described previously (21). In the analysis of p21^WAF1/CIP1 and p16^INK4a protein, rabbit p21^WAF1/CIP1polyclonal antibody sc-397, mouse p16^INK4a monoclonal antibody sc-1661 (Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-rabbit IgG peroxidase conjugate (ICN Biomedicals, Costa Mesa, CA) and rabbit anti-mouse IgG peroxidase conjugate (Santa Cruz Biotechnology) were used as primary and secondary antibodies.

Flow cytometry
For each sample, ~10⁶ cells were washed with ice-cold PBS and fixed in 95% ethanol. Cells were then resuspended in 1 mg/ml RNase (Sigma) for 30 min at 37°C and stained with 0.05 mg/ml propidium iodide (Sigma) for 1 h on ice. Flow cytometric analysis was performed with a FACS Vantage flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Cells were excited at 488 nm and the emission was detected through a 630 ± 11 nm band pass filter. A minimum of 10 000 cells were analyzed for each sample. Cell-cycle analysis was performed using Modfit 5.2 software (Verity Software House, Topsham, ME).

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
We first investigated the cell-cycle distribution of NAC-treated 308 cells. As shown in Table I, the cells accumulated in G₀/G₁ phase within 48 h, with an accompanying decrease of cells in both S phase and G₂/M phase. This G₁ cell-cycle arrest was significant at an NAC dose of 5 mM. Lower levels of G₀/G₁ accumulation could be observed at a dose of 1 mM. This observation was reproduced in the BALB/c 3T3 A31 fibroblast cell line (data not shown), indicating that this arrest by NAC is not cell-type specific.

View this table: [in this window] [in a new window]   Table I. NAC-induced changes in the percentage of 308 cells in each stage of the cell cycle.

 
Cell-cycle progression is precisely regulated by a series of cyclins, cdks and cdk inhibitors. Given the known functions of cdk inhibitors p21^WAF1/CIP1 and p16^INK4a in controlling cell-cycle progression at G₁, we next examined the expression of both genes in NAC-treated cells. As shown in Figure 1A, NAC treatment of 308 cells resulted in 3- to 5-fold induction of p21^WAF1/CIP1 protein and mRNA. Surprisingly, p21^WAF1/CIP1 protein elevation by NAC was also detected in the p53-deficient (10)1 fibroblast cell line (Figure 1A). These observations indicate that p21^WAF1/CIP1 induction by NAC is independent of p53. Moreover, the G₁-specific cdk inhibitor p16^INK4a was also induced by NAC (Figure 1B). p16^INK4a is a specific inhibitor of cyclin D-dependent kinase, and it is believed to mediate growth suppression by increasing hypophosphorylated pRb, which in turn leads to G₁ cell-cycle arrest (22).

View larger version (41K): [in this window] [in a new window]   Fig. 1. (A) p53 independence of p21WAF1/CIP1 induction by NAC. Western blot analysis of p21WAF1/CIP1 protein induction by 20 mM NAC in 308 cells (upper); northern blot of p21WAF1/CIP1 mRNA in 308 cells after 5 h exposure to NAC (middle); western blot analysis of p21WAF1/CIP1 protein expression after treatment with 20 mM NAC in p53-deficient 10(1) cells (lower). (B) p16INK4a protein induction by 5 mM NAC in 308 cells. Data shown represent similar results from one of two experiments.

 
These studies show that NAC upregulates the expression of p21^WAF1/CIP1 and p16^INK4a. To investigate the contribution of these proteins, as well as p53, to the G₁ cell-cycle arrest by NAC, we performed genetic analysis by using gene-null cells. As shown in Figure 2A and B and Table II, while p53-null and p21^WAF1/CIP1-null cells retained the full G₁ arrest, ~40% of the arrest was lost in p16^INK4a knockout cells. Therefore, we conclude that the G₁ arrest by NAC is partially governed by p16^INK4a, but not by p21^WAF1/CIP1 or p53.

View larger version (30K): [in this window] [in a new window]   Fig. 2. p53 independence of G1 arrest by NAC. (A) Profiles of cell-cycle distribution in p53-deficient (10)1 cells treated with NAC for 24 h. (B) Profiles of cell-cycle distribution in mouse keratinocyte cell line NHK-4 (p53–/–) treated with 5 mM NAC for 24 h. Data shown represent similar results from one of two or three experiments.

 

View this table: [in this window] [in a new window]   Table II. Effects of 24 h treatment of 5 mM NAC on mouse primary keratinocytes from different genotypes

 
We next investigated whether NAC-induced G₁ phase delay could be reversed by removing NAC from the medium. 308 cells were treated with 20 mM NAC for 24 h, and the medium was replaced with medium not containing NAC. As seen in Figure 3, 24 h after the medium replacement, cells resumed their regular cell-cycle distribution. Thus, the NAC effect on G₁ arrest is reversible.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Reversibility of G1 arrest by NAC. 308 cells were incubated with medium containing 20 mM NAC for 24 h, and replaced with regular medium containing no NAC. Twenty-four hours later, cells were subjected to flow cytometry analysis. Data shown represent similar results from one of two experiments.

 
A major intracellular pathway of NAC metabolism is deacetylation to the thiol cysteine, and subsequent incorporation into GSH (13). GSH is the major cellular antioxidant. To test the possibility that NAC acts by increasing the intracellular GSH level, we pre-treated and co-incubated cells with L-buthionine sulfoximine (BSO), a potent inhibitor of GSH synthesis. Significantly, G₁ arrest by NAC was not blocked by 20 µM BSO (Figure 4). This dose of BSO was found to completely block the increase in cellular GSH level after NAC (data not shown). BSO itself has little effect on cell-cycle distribution (Figure 4). These results imply that NAC prolongs the G₁ transition via a GSH-independent mechanism.

View larger version (14K): [in this window] [in a new window]   Fig. 4 Independence of GSH in G1 arrest by NAC. Cell cycle distributions were analyzed in 308 cells treated with BSO and/or NAC. Cells treated with both compounds were preincubated with medium containing 20 µM BSO for 1 h and then treated with medium containing 20 µM BSO and 5 mM NAC for 24 h. Data shown represent similar results from one of two experiments.

 
Since NAC is an antioxidant, we also tested a chain-breaking antioxidant, Trolox, which is a water-soluble analog of Vitamin E. At a dose of 1 mM (the maximal solubility in medium), Trolox did not alter the cell cycle-distribution or cause any G₁ arrest (Figure 5). This result indicates that the G₁-phase accumulation is not a general effect of antioxidants and suggests, but does not prove, that the cell-cycle arrest by NAC is mediated by its redox effect rather than free radical scavenging.

View larger version (13K): [in this window] [in a new window]   Fig. 5. No effect of non-sulfur containing antioxidant Trolox on cell cycle progression. 308 cells were incubated with 1 mM Trolox for 24 h, and then subjected to flow cytometry analysis. Data shown represent similar results from one of two experiments.

 
All of the above studies were performed in mouse cells. In order to assess the possibility that G₁-phase prolongation may contribute to NAC's chemoprevention of human cancers, we next investigated whether NAC could delay G₁ transition in human cells. Human primary foreskin fibroblasts were treated with 1 mM NAC for 24 h, and subjected to flow cytometry analysis. As shown in Figure 6, G₁ arrest indeed occurred in normal human cells. This observation indicates that G₁ arrest by NAC is not species specific.

View larger version (15K): [in this window] [in a new window]   Fig. 6. G1 arrest by NAC in human primary foreskin fibroblasts. Fibroblasts were incubated with 1 mM NAC for 24 h, and then subjected to flow cytometry analysis. Data shown represent similar results from one of two experiments.

 

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
In this report, we show that NAC treatment upregulates p21^WAF1/CIP1 and p16^INK4a expression, and prolongs the G₁-phase transition in part via p16^INK4a. G₁ arrest by NAC is independent of p53 and p21^WAF1/CIP1. As a chemopreventive agent, NAC has often been reported to exert its chemopreventive activities by blocking the formation of DNA–carcinogen adducts, either directly or indirectly (5). Our current findings unveil a potential mechanism by which NAC may accomplish its chemoprevention function in normal cells by inducing p16^INK4a-mediated G₁ cell-cycle arrest and p21^WAF1/CIP1 gene expression.

As an antioxidant, NAC was demonstrated to inhibit DNA synthesis by ras-transformed fibroblasts, via scavenging reactive oxygen species elicited by activated ras (23). However, the role of reactive oxygen species in normal cells is less clear, since these were not measured (23). If normal mitogenic signaling is mediated by reactive oxygen species, our data suggest that reactive oxygen species act partially by inhibiting p16^INK4a and thus relieving G₁ prolongation.

In view of our previous report on NAC-induced apoptosis (14), and results presented in this study, it is clear that the fate of 308 papilloma cells after NAC treatment can be modulated by the dose applied. At a dose of 5 mM or lower, G₁ arrest occurs with very little apoptosis; at 20 mM, both G₁ arrest and apoptosis are induced. In contrast to G₁ arrest, however, the apoptosis by NAC is not detected in p53-deficient cells (14). These findings suggest that G₁ arrest and apoptosis induced by NAC occur through different pathways, with the G₁ arrest acting through a p53-independent mechanism.

In their lifetimes, humans are constantly exposed to low doses of a variety of environmental carcinogens. Since it is impossible to design different chemopreventive agents to inhibit innumerable carcinogens with distinctive mechanisms, it is reasonable to make the hypothesis that one effective strategy for chemoprevention would be to apply extrinsic regulators that facilitate natural cellular defenses against general genetic insults. Activation of tumor suppressor genes, such as p16^INK4a and p53 (22,24), fits well with this strategy. Cell proliferation is a risk factor for response to carcinogenic stimuli, such as DNA damage. By slowing down cell proliferation via p16^INK4a induction, NAC may facilitate cellular machinery for repairing genomic damage by carcinogens before cell division.

While tumor suppressor genes such as p16^INK4a and p53 would function well in normal cells, they can be inactivated through gene alterations during tumorigenesis. Their lost functions may be offset by other genes with anti-tumorigenesis activities, such as p21^WAF1/CIP1. Unlike p16^INK4a and p53, there are few p21^WAF1/CIP1 gene mutations detected in human malignancy (25). p21^WAF1/CIP1 has been suggested to play a role in DNA repair and slowdown of DNA replication in S phase (26–28), in addition to cell cycle control (19). Therefore, p53-independent p21^WAF1/CIP1 activation by NAC could function in both normal and precancer cells.

	Acknowledgments

 
We are very grateful to Drs P.Leder, M.Serrano, W.Weinberg and A.Levine for providing transgenic mice and cell lines, respectively. This work was supported by NIH grant CA55737 (D.E.B.) and the Leslie H.Warner Postdoctoral Fellowship in Cancer Research (to M.L.).

	Notes

 
³ To whom correspondence should be addressed Email: douglas.brash{at}yale.edu

	References

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Received June 5, 1998; revised May 5, 1999; accepted May 14, 1999.

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