5-Aza-2'-deoxycytidine is chemopreventive in a 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone-induced primary mouse lung tumor model

Laura E. Lantry1, Zhongqiu Zhang1, Keith A. Crist2, Yian Wang1, Gary J. Kelloff3, Ronald A. Lubet3 and Ming You1,4

1 Departments of Pathology and
2 Surgery, Medical College of Ohio, Health Education Building, Room 202, Toledo, OH 43614 and
3 Chemoprevention Branch, National Cancer Institute, Rockville, MD 20892, USA


    Abstract
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 Abstract
 Introduction
 References
 
Carcinogenesis is a multistep process in which many alterations in both genetic and epigenetic controls lead to a growth advantage for neoplastic cells. Hypermethylation has been established as the basis of genomic imprinting, but recent studies have also shown that alterations in genomic methylation patterns may contribute to tumorigenesis. The chemical 5-aza-2'-deoxycytidine (5-aza-dC) has been used both in vitro and in vivo to inhibit DNA methylation. In this study, we investigated the chemopreventive efficacy of 5-aza-dC in a well-established primary mouse lung tumor model. Five-week-old male (C3H/HeJxA/J) F1 hybrid mice were treated for 24 consecutive weeks with 5-aza-dC, three times per week i.p. Lung tumors were induced with two consecutive weekly doses of 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone starting 1 week after initial treatment with 5-aza-dC. We demonstrated that 5-aza-dC exhibits a chemopreventive effect in this primary mouse lung tumor model which, like human lung adenocarcinomas, harbors an activating K-ras mutation. Treatment with 5-aza-dC resulted in a 23% reduction in tumor incidence, as well as a 42% reduction in tumor multiplicity. This work supports further investigation of methylation inhibitors likes 5-aza-dC for early intervention, prevention and treatment of lung cancer.

Abbreviations: 5-aza-dC, 5-aza-2'-deoxycytidine; C3A F1, (C3H/HeJxA/J) F1; LOH, loss of heterozygosity; MTase, methyltransferase; MTD, maximum tolerated dose; NNK, 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone; PBS, phosphate-buffered saline.


    Introduction
 Top
 Abstract
 Introduction
 References
 
Carcinogenesis evolves in a complex, multistep milieu of both genetic and epigenetic changes. Aberrant methylation, including hypomethylation resulting in activation of protooncogenes, and hypermethylation, reported to silence tumor suppressor genes, have been suggested to contribute to this process by altering homeostasis, giving a growth advantage to initiated cells over the neighboring normal population (1). These alterations may lead to an ever-increasing state of genomic instability, potentially culminating in malignancy (1).

Aberrant patterns of methylation including both hypomethylation and hypermethylation have been associated with many human tumors (24). Inactivation of the p16INK4a tumor suppressor gene has been shown to result from de novo methylation of the 5'-promoter region in the absence of loss of heterozygosity (LOH) (57). In lung cancer, investigators have shown both DNA hypermethylation resulting in silencing of the p16INK4a gene (8), as well as an increase in DNA methyltransferase (MTase) activity (9). In colon cancer, overexpression of the DNA MTase gene was found to precede changes in the pattern of methylation and increased with progression of the disease (4). Based on these results, Baylin (10) proposed that, in solid tumors, a pattern of methylation imbalance begins with an increase in methylation capacity at the hyperplasia stage of tumorigenesis. The underlying theme inherent in this hypothesis is that changes in methylation patterns are capable of altering chromatin structure, which in turn directly or indirectly alters gene expression by transcriptional activation or inactivation. Furthermore, this process contributes to the increased level of genetic instability seen with progression of cancer (10).

The chemical 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methylation inhibitor, forms a covalent bond between the DNA and the enzyme DNA MTase, thus effectively inactivating the enzyme (11). At low concentrations, 5-aza-dC has been shown to reactivate silenced genes in vitro (6,8). In a recent study, Bender et al. investigated the nature of the inhibition of immortalized tumor cell line growth in response to 5-aza-dC and found that such treatment upregulated the expression of the p16INK4a gene by demethylating the 5' promoter region (12). In human breast cancer cells, 5-aza-dC was shown to act both by demethylating the estrogen receptor gene and by forming stable DNA MTase–DNA adducts which induced apoptosis in some cell lines (13). In addition, 5-aza-dC has been used in chemotherapeutic clinical trials for leukemia and some solid tumors (14,15).

Recently, Laird et al. (16) reported that administration of 5-aza-dC to Min mice resulted in >98% inhibition of ApcMin-induced intestinal neoplasia, presumably due to the reduction of DNA MTase activity. In the present study, we demonstrate that 5-aza-dC is chemopreventive in a mouse lung tumor model when administered prior to chemical initiation, and throughout the promotion phase of mouse lung tumorigenesis.

4-(Methyl-nitrosamino)-1-(3-pyridyl-1-butanone (NNK) (99% pure) was purchased from Chemsyn Science Laboratories (Lenexa, KS). NNK was prepared in warmed phosphate-buffered saline (PBS). 5-Aza-dC (>99%) was purchased from Sigma (St Louis, MO). (C3H/HeJ x A/J) F1 (C3A F1) and A/J mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed four per cage, in plastic cages with hardwood bedding and dust covers, in an HEPA-filtered, environmentally controlled room (24 ± 1°C, 12/12 h light/dark cycle). Animals were given Rodent Lab Chow, No. 5001 (Purina) and water ad libitum. Following a 7 day quarantine, the animals were randomized into treatment groups, after which weights were monitored weekly for the duration of the study.

The i.p. maximum tolerated dose (MTD) for 5-aza-dC was determined prior to the beginning of the lung tumor bioassay. For the purposes of this study, the MTD was defined as the dose at which no animals died as the result of treatment and at which overt signs of toxicity were absent, including, but not limited to, >10% loss of body weight, abdominal edema, severe hair loss or paralysis/weakness. The MTD study was conducted over a period of 10 weeks, which included 6 weeks of active injection, and 4 weeks of observation. The 5-aza-dC was tested at 1, 2, 5 and 10 mg/kg.

The compound solubilized readily in PBS at the stock concentration of 2.5 mg/ml. The 4-week-old male A/J mice were randomized into four treatment groups (n = 4/group) and given three i.p. injections per week of the appropriate concentration of 5-aza-dC (0.1 ml/injection) for the duration of the 6 week period. During the 4 week observation period the mice were monitored for overt symptoms of toxicity as stated above. The mice were weighed throughout the MTD study. The animals were killed by CO2 asphyxiation and then inspected for non-overt signs of toxicity, including edema, enlargement or atrophy of internal organs (liver, spleen, kidneys, heart).

The results of the MTD study were as follows: 5-aza-dC showed toxicity at the three highest doses (2 mg/kg, 5 mg/kg and 10 mg/kg). Treatment was lethal for one mouse in the highest dose group (10 mg/kg), detected after the seventh i.p. dose (total i.p. doses = 18); for one mouse in the 5 mg/kg group at the ninth i.p. dose; and for one mouse in the 2 mg/kg group after the 13th i.p. In addition, body weights of the mice in the two highest dose groups (5 and 10 mg/kg) did not increase commensurate with age. Based on this 10 week study, the 1 mg/kg dose was set as the MTD for the long-term bioassay, based on absence of treatment-associated lethality or overt symptoms of toxicity.

The lung tumor bioassay was designed to evaluate the chemopreventive efficacy of 5-aza-dC prior to initiation and throughout the promotion phase of lung tumorigenesis. Based on the results of the MTD study, evaluation of 5-aza-dC for chemopreventive efficacy began at 1 mg/kg, in the NNK-induced C3A F1 mouse lung tumor model. Starting 1 week prior to NNK treatment, the compound was delivered i.p. in a total volume of 0.1 ml, three times per week for 24 weeks at the MTD for the initial 12 weeks, and, due to latent toxicity encountered during the bioassay, at 0.5 MTD for the last 12 weeks. Five-week-old male C3A F1 hybrid mice were randomized into groups as follows: group 1, 5-aza-dC + NNK (n = 16); group 2, 5-aza-dC (n = 16); group 3, NNK control (n = 10); group 4, vehicle control (n = 10). NNK (100 mg/kg) was given i.p. once per week for 2 weeks, 24 h after the fourth and seventh doses of 5-aza-dC. The mice were monitored for signs of toxicity on a weekly basis (weight loss, loss of or roughened appearance of fur, abdominal edema, etc.). Animals were killed by CO2 asphyxiation at week 24. The fresh tissue was inspected for lung tumors, with up to three tumors per lung harvested and flash frozen in liquid nitrogen for future analysis. The lungs were fixed in 10% buffered formalin overnight, and then kept in ethanol until paraffin embedding. Each lung was examined by at least two investigators under a dissecting microscope to obtain the surface tumor count, which was added to the harvested tumor number to obtain the total tumor count. Selected blocks were sectioned at 5 µm, stained by H & E and examined by light microscopy to confirm tumor histology. Unpaired Student's t-tests were run to test for significant statistical difference.

Treatment with 5-aza-dC reduced the incidence of tumors in group 1 by 23% compared with control (group 3). Furthermore, lung tumor multiplicity was reduced by 42% below control, with a mean of 1.38 ± 0.96 tumors/mouse (P < 0.05) as shown in Table IGo. There were three mice in group 2 with one tumor each; however, this was not significantly different from the control group. Although we chose, based on the preliminary toxicity assay, a dose of 5-aza-dC that was not toxic, three animals in group 1 and two animals in group 2 died by the 12th week of treatment. Therefore, the dose of 5-aza-dC was lowered to one half of the MTD (0.5 mg/kg) for the remaining 12 weeks of the study. The remaining animals recovered from overt signs of toxicity within 1–2 weeks. As seen in the Table IGo, regardless of treatment regimen, the average body weight of mice at the end of the study was not significantly different between groups. The lung tumors were histologically confirmed as adenomas by light microscopy of H & E stained sections. The results from this study indicate that 5-aza-dC prevents tumor formation in a primary mouse lung tumor model.


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Table I. Effect of 5-aza-2'-deoxycytidine on NNK-induced lung tumorigenesis in male C3A F1 hybrid micea
 
Previous studies have demonstrated that DNA methylation plays an important role in the carcinogenic process through several mechanisms, including mutation potential at 5-methyl-dC, regional hypermethylation, global hypomethylation and increased expression of DNA MTase (14). Methylated cytosine can undergo spontaneous deamination to thymine, which is repaired by DNA mismatch repair mechanisms, but with only ~90% efficiency (17). The repair mechanism itself has been identified as a target in tumorigenesis (3,18). Regional hypermethylation may silence genes controlling cell cycle progression, as seen in the p16INK4a gene (6,12). An effect of altered methylation patterns may be seen in the conformation of the DNA. Hypermethylation in CG rich `hotspots' may stabilize left-handed Z-DNA under physiologic conditions (19). Hypermethylation has also been implicated in the formation of human lung and colon tumors (20). Widespread hypomethylation may alter the structure of chromatin, increasing the likelihood of instability and/or interaction with DNA regulatory proteins (10,21). Moreover, altered DNA methylation patterns have been associated with the progression of some solid human tumors (24).

The most probable mechanism for the observed chemopreventive effect of 5-aza-dC against NNK-induced mouse lung tumorigenesis is the activation of hypermethylated tumor suppressor genes. Hypermethylation of tumor suppressor genes and differentiation-specific genes has frequently been seen in cancer cells, presumably due to the increased expression of DNA MTase (14). For example, the p16INK4a gene has been shown to be inactivated by hypermethylation in several cancer types and treatment with 5-aza-dC could reactivate the gene, suggesting that 5-aza-dC may inhibit the tumor cells by reactivating the p16INK4a gene and/or other tumor suppressor genes silenced by hypermethylation (5,2230). Interestingly, p16INK4a inactivation, either by allelic deletion or hypermethylation, is a frequent genetic defect in lung tumor progression, as demonstrated in studies using immunohistochemical analysis (3134). Markedly reduced or absent p16 protein was shown in approximately half of the A/J and C3A F1 lung adenocarcinomas, some of which revealed only focal areas of loss (34; Q.Liu et al., unpublished data). Thus, there is compelling evidence that the regulatory regions of the p16INK4a gene could become hypermethylated with tumorigenesis, and it has been proposed that this may set the stage for the mechanism by which LOH occurs during the progression of disease to malignancy. Consistent with our results in the mouse lung tumor model, Laird et al. (16) have reported that 5-aza-dC reduced the average number of intestinal adenomas from 113 in the control group to two in the treatment group in the ApcMin mouse model. That study, together with our results from the present study, provide further evidence that alterations in the pattern of DNA methylation play a key role in the process of carcinogenesis and that inhibition of DNA methylation may prevent or slow the process of tumorigenesis.

Cytotoxic levels of 5-aza-dC are already being utilized in some chemotherapeutic settings. Our results show that it may also be useful at earlier stages of tumorigenesis and may indeed have efficacy in the reduction of neoplasia subsequent to traditional chemical and/or surgical therapies for lung cancer. Caution is warranted as this chemical has also been implicated in an increase in tumorigenesis due to inherent mutagenicity, as well as in increases in cellular transformation of BALB/c-3T3 cells (35,36). Consistent with these observations, we have seen an increase in both lung tumor incidence (23%) and multiplicity (0.23 tumors/lung) in mice treated with 5-aza-dC (Table IGo, group 2) as compared with vehicle control mice (Table IGo, group 4). In summary, these data support further investigation of methylation inhibitors like 5-aza-dC for early intervention, prevention and treatment of lung cancer.


    Acknowledgments
 
We thank Drs Gary D.Stoner and Herman A.J.Schut for critical reading of the manuscript. We would also like to acknowledge Drs Feng Gao, Ping Gu and Mark Morse for excellent technical assistance. This work was supported by NIH grants CA58554 and CN55184.


    Notes
 
4 To whom correspondence should be addressed Email: myou{at}mco.edu Back


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Received July 31, 1998; revised September 22, 1998; accepted October 9, 1998.