Elsevier

Life Sciences

Volume 71, Issue 4, 14 June 2002, Pages 421-436
Life Sciences

Induction of apoptosis and inhibition of papilloma formation may signal a new role for okadaic acid

https://doi.org/10.1016/S0024-3205(02)01701-0Get rights and content

Abstract

Okadaic acid (OA), a tumor promoter in the mouse skin carcinogenesis model, has been shown to induce apoptosis in tumor cell lines that harbor H-ras mutations. We examined the effects of OA on mouse keratinocytes with (308) and without (C50) H-ras mutation in vitro and in an in vivo system. Following exposure to varying concentrations of OA over time, the effects of OA in vitro were assessed using microscopic, biochemical and flow cytometric techniques. OA effects on the cells included incorporation of propidium iodide, externalization of phosphatidylserine, and development of hypodiploidy. 308 cells demonstrated typical DNA ladder formation, rapid chromatin and nuclear condensation, while C50 cells demonstrated delayed chromatin condensation and nuclear fragmentation, but no DNA ladder formation. In vivo, OA elicited delayed papilloma formation and reduced tumor multiplicity. Though its mechanism of action is not fully known, we found that OA-induced inhibition of the clonal expansion of initiated cells may be related to the presence or absence of H-ras mutation.

Introduction

In the multi-step mouse skin carcinogenesis model, initiation of mouse skin with 7,12-dimethylbenz[a]anthracene (DMBA), followed by promotion with one of several tumor promoters resulted in the formation of skin papillomas [1], [2], [3]. Okadaic acid (OA), a toxic polyether compound, first isolated from marine sponges [4], has been reported to be a potent non-phorbol promoter of papilloma formation in the DMBA-induced mouse skin carcinogenesis model [3], [5]. Fujiki et al. [3] and Nelson et al. [6] reported that initiation of mouse skin with DMBA produced mutation in codon 61 of the Harvey ras gene (H-ras). OA is an inhibitor of protein phosphatases 1 and 2A [7], [8], [9], and an inducer of apoptosis in multiple cell lines [10], [11], [12].

We find reports that OA serves as a tumor promoter in intact mouse skin but an inducer of apoptosis in cells with ras mutation in vitro, to be inconsistent. To investigate the relationship between OA, apoptosis induction and skin tumor promotion, we studied the effects of OA on mouse keratinocyte cell lines (308 and C50) in culture and in an in vivo mouse skin carcinogenesis model. Both cell lines have been used to study skin carcinogenesis process. 308 cells contain an H-ras mutation and produced papillomas when grafted on athymic nude mice and promoted with TPA [13], [14]. C50 cells are immortalized cells that harbor wild-type H-ras and did not form papillomas when grafted on athymic nude mice and promoted with TPA [13].

In this report, we present results showing that upon exposure to OA, mouse keratinocytes 308 cells (with mutation in the H-ras gene) exhibited early signs of apoptosis while C50 cells (with wild-type H-ras gene) were less sensitive. We also found that treatment of initiated cells with OA elicited a reduction in the tumor multiplicity in mice initiated with DMBA and promoted with TPA. Although the mechanism(s) of apoptosis induction in these cells is not yet known, our data suggest that OA induced cell death in mouse keratinocytes in vitro and in initiated cells in vivo.

Section snippets

Reagents and media

Okadaic acid was first a gift from Dr. Hirota Fujiki (Saitama Cancer Center Research Institute, Saitama, Japan) and later purchased from Alexis Biochemicals (San Diego, CA). Cell dissociation solution, ribonuclease A (RNase A) and dimethylsulfoxide (DMSO) were obtained from Sigma (St. Louis, MO). Minimum essential medium (S-MEM, lacking Ca2+ and Mg2+), penicillin (100 units/ml) and 100μg/ml streptomycin (P/S), fetal bovine serum (FBS) and L-glutamine were obtained from GIBCO (Grand Island, NY).

Cell culture

OA induced detachment of cells from the substratum

The initial response of keratinocytes growing in monolayers to OA treatment was loss of adhesion and detachment from the substratum. Loss of attachment to the substratum by adherent cells has been reported as an early observable event in the cell death process [26]. Cells treated with 20 nM OA exhibited loss of adhesion as early as twelve hours (data not shown). Phase-contrast microscopy (Fig. 1) illustrates this process in C50 and 308 cells.

To study the effect of OA on cell proliferation, we

Discussion

We set out to investigate the effects of OA on mouse keratinocytes. We used mouse keratinocyte cell lines which have been shown to behave similarly to mouse skin initiated with DMBA and promoted with TPA in the multi-step mouse skin carcinogenesis model. Our results indicate that OA caused differential cell death in mouse keratinocyte cell lines C50 and 308. Phase-contrast microscopy showed that when exposed to OA, the cells lost adhesion and detached from the substratum (Fig. 1). When cells

Acknowledgments

We thank Dr. Steve Carper for helpful discussions during the preparation of this manuscript. This work was supported by NIH Grant CA 35368S1 (Research Supplement for Underrepresented Minorities to JAE), NIH Grant CA 35368, NIH core grant CA 14520-26, and the Veterans Administration Research Service.

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