LETTERS TO THE EDITOR

The p53-Stabilizing Compound CP-31398 EnhancesUltraviolet-B-Induced Apoptosis in a Human MelanomaCell Line MMRU

To the Editor:

The tumor suppressor p53 plays a crucial role in cellularstress response to ultraviolet (UV) irradiation, including cellcycle arrest, DNA repair, and apoptosis. As the p53 proteinlevel in unstressed cells is very low due to a short half-life,compounds that stabilize the p53 protein conformation mayprolong its tumor suppressive functions. Recently, a syntheticcompound, CP-31398, was found to promote the stability ofthe DNA binding domain of wild-type p53 and full-lengthwild-type p53 (Foster et al, 1999). CP-31398 was also able to stabi-lize the active conformation of newly synthesized mutant p53,leading to increased transcriptional activity and inhibition of tu-mor growth in mice.We recently showed that CP-31398 inducedp53-dependent apoptosis in human colon carcinoma cellsthrough the Bax/mitochondrial/caspase-9 pathway (Luu et al,2002). In this communication, we show that CP-31398 signi¢-cantly enhances UVB-induced apoptosis in a human melanomacell line.To determine if CP-31398 could upregulate p53 protein

levels after UVB exposure, a wild-type p53 human melanomaline, MMRU, was exposed to UVB and treated with CP-31398.Western analysis showed that p53 protein levels increased 2.1-and 2.4-fold after treatment with CP-31398 or UVB alone,respectively, compared to the untreated control (Fig 1A). Combi-nation treatment with UVB and CP-31398, however, led to ahigher p53 induction (4.8-fold) than either UVB or CP-31398treatment alone. To determine if CP-31398 enhances UVB-induced apoptosis, propidium iodide and £ow cytometry analysiswas performed to assess the pre-G1 population. UVB-irradiatedcells treated with CP-31398 had a signi¢cantly higherpercentage of hypodiploid DNA population than cells treatedwith UVB alone (Fig 1B). Treatment with CP-31398 alone at8 mg per ml is nontoxic to the cells. In addition, the DNA ladderassay showed more DNA fragmentation in UVB-irradiated cellstreated with CP-31398 compared to cells that were only irradiatedwith UVB (Fig 1C). Collectively, these data show that CP-31398enhances UVB-induced apoptosis in human melanoma MMRUcells.Next, we sought to elucidate the mechanism involved in CP-

31398 enhancement of UVB-induced apoptosis.Western analysisshowed that Bax, a pro-apoptotic protein known to be regulatedby wild-type p53 and to be involved in UV-induced cell death,was upregulated more in the UVB/CP-31398 combination treat-ment than the single treatments (Fig 2A). Increased Bax expres-sion can result in mitochondrial membrane permeability changeand release of cytochrome c from the mitochondria into the cy-toplasm (Karpinich et al, 2002).We then used the MitoCaptureTM

Apoptosis Detection Kit to determine if CP-31398 has an e¡ecton mitochondrial membrane potential. There was a signi¢cantlyhigher percentage of green apoptotic cells in UVB-irradiatedcells treated with CP-31398 compared to the cells irradiatedwith UVB alone (49% vs 33% at 40 mJ per cm2, po0.05; 55%vs 40% at 60 mJ per cm2, po0.05, Student t test) (Fig 2B),suggesting that CP-31398 enhances UVB-induced apoptosis byaltering the mitochondrial membrane potential. Subcellularfractionation and Western blotting indicated that more cyto-chrome c was released from the mitochondria in MMRU cellsafter UVB/CP-31398 combination treatment whereas little cyto-chrome c release occurred in cells treated with UVB or CP-31398alone (Fig 2A).Activation of the mitochondrial apoptotic pathway involves

cytochrome c release, leading to the activation of caspase-9 andsubsequent downstream e¡ector caspases, such as caspase-3 (ShenandWhite, 2001).We analyzed the cleavage of caspase-9 and cas-pase-3 byWestern blotting. More procaspase-9 and procaspase-3were cleaved in UVB-irradiated cells treated with CP-31398 com-pared to cells irradiated with UVB alone (Fig 2C). Collectively,these data show that CP-31398 activates the mitochondrial apop-totic pathway after UVB exposure.Our results for the ¢rst time demonstrate that CP-31398

enhances UVB-induced apoptosis in human melanoma cellsvia the Bax/mitochondria/caspase-9 pathway. These results areconsistent with previous studies showing the involvement ofp53 in the mitochondrial-mediated apoptotic pathway. Studieshave shown that UV exposure of mouse skin induced p53expression followed by increased Bax expression and apoptosis(Ouhtit et al, 2000). Moreover, adenoviral introduction of wild-type p53 into p53-null Saos-2 cells causes Bax-dependent cyto-chrome c release and activation of caspases (Schuler et al, 2000).p53-dependent apoptosis is a self-protective mechanism to elim-inate unnecessary cell proliferation or delete cells with severelydamaged DNA. Failure to remove cells with damaged DNA byapoptosis may lead to mutations and ultimately cancer develop-ment. Thus, the presence of wild-type functional p53 is impor-tant to the preservation of genomic integrity and prevention ofcarcinogenesis after UV exposure. p53 has been shown to beinvolved in UV-induced skin cancer development in animalmodels. Transgenic mice with abnormal p53 function developmore squamous cell carcinomas than control mice after UVBirradiation (Li et al, 1995; 1998). Furthermore, Kanjilal et al (1993)found that all 11 of UV-induced murine skin cancers examinedhad mutations in the p53 gene. In humans, individuals withLi^Fraumeni syndrome who have germ-line mutations in thep53 gene are highly susceptible to the development of manyforms of cancer (Nichols et al, 2001). Our ¢ndings of CP-31398enhancement of UVB-induced apoptosis in melanoma cellssuggest that this compound may be used to stabilize p53 or rescuemutant p53 to suppress the development of skin cancer after UVexposure. More studies are required to determine if CP-31398 canenhance UVB-induced apoptosis in normal nontransformedmelanocytes and keratinocytes.

Reprint requests to: Dr. Gang Li, Jack Bell Research Center, 2660 OakStreet,Vancouver, BC, CanadaV6H 3Z6; Email: gangli@interchange.ubc.caAbbreviations: UV, ultraviolet.

Manuscript received June 25, 2002; revised July 24, 2002; accepted forpublication August 5, 2002

0022-202X/02/$15.00 � Copyrightr 2002 by The Society for Investigative Dermatology, Inc.

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We thank P¢zer Inc. for providing the CP-31398 compound.This study was sup-ported in part by the Canadian Dermatology Foundation and the Canadian Instituteof Health Research. Dr. Gang Li is a recipient of a Research Scientist Award from theNational Cancer Institute of Canada supported with funds provided by the CanadianCancer Society.

Yvonne Luu and Gang LiDepartment of Medicine, Division of Dermatology,

Vancouver Hospital and Health Sciences Center,University of British Columbia,Vancouver, Canada

REFERENCES

Foster BA, Co¡ey HA, Morin MJ, Rastinejad F: Pharmacological rescue of mutantp53 conformation and function. Science 286:2507^2510, 1999

Kanjilal S, Pierceall WE, Cummings KK, Kripke ML, Ananthaswamy HN: Highfrequency of p53 mutations in ultraviolet radiation-induced murine skintumors: evidence for strand bias and tumor heterogeneity. Cancer Res 53:2961^2964, 1993

Figure 2. CP-31398 activates the mitochondrial pathway duringUVB-induced apoptosis. MMRU cells were irradiated with 0 or 40 mJper cm2 of UVB followed by treatment with 0, 8, or 10 mg per ml of CP-31398 for 24 h. (A) Western analysis of Bax and cytochrome c levels withanti-Bax (N-20) (Santa Cruz Biotechnology) and anticytochrome c (BDPharmingen) antibodies. (B) Cells were stained with MitoCaptureTM solu-tion containing a cationic dye and then visualized using £uorescent micro-scopy. The graph shows quanti¢cation of the percentage of green apoptoticcells based on the changes in the mitochondrial membrane potential. Atleast 500 cells from ¢ve random ¢elds were counted. Data representmean7SD from three independent experiments. Paired two-tailed Stu-dent’ t test. nSigni¢cance: po0.05, compared with UVB alone. (C)Westernanalysis of caspase-9 and caspase-3 levels. b-actin was used as loading con-trol. The relative expression levels of procaspase-9 and procaspase-3 weredetermined by densitometry.

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Figure1. CP-31398 enhances UVB-induced apoptosis by increasingp53 protein levels in MMRU cells.MMRU cells were irradiated with 0,40, 60, or 80 mJ per cm2 of UVB followed by treatment with 0 or 8 mg perml of CP-31398 for 24 h. (A) Western analysis of p53 level with DO-1antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and b-actin (BDPharmingen, Canada) as loading control. The fold of p53 induction wasdetermined by densitometry. (B) Cells were stained with propidium iodideand the pre-G1 population was determined by £ow cytometry. (C) DNAwas extracted and analyzed on a 2% agarose gel.

1208 LETTERS TO THE EDITOR THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Karpinich NO,Tafani M, Rothman RJ, Russo MA, Farber JL: The course of etopo-side-induced apoptosis from damage to DNA and p53 activation to mitochon-drial release of cytochrome c. J Biol Chem 277:16547^16552, 2002

Li G, Ho VC, Berean K, Tron VA: Ultraviolet radiation induction of squamous cellcarcinomas in p53 transgenic mice. Cancer Res 55:2070^2074, 1995

Li G,TronV, HoV: Induction of squamous cell carcinoma in p53-de¢cient mice afterultraviolet radiation. J Invest Dermatol 110:72^75, 1998

Luu Y, Bush J, Cheung KJ, Li G: The p53 stabilizing compound CP-31398 inducesapoptosis by activating the intrinsic Bax/mitochondrial/caspase-9 pathway. ExpCell Res 276:214^222, 2002

Nichols KE, Malkin D, Garber JE, Fraumeni JF Jr, Li FP: Germ-line p53 mutationspredispose to a wide spectrum of early-onset cancers. Cancer Epidemiol Biomar-kers Prev 10:83^87, 2001

Ouhtit A, Muller HK, Davis DW, Ullrich SE, McConkey D, Ananthaswamy HN:Temporal events in skin injury and the early adaptive responses in ultraviolet-irradiated mouse skin. AmJ Pathol 156:201^207, 2000

Schuler M, Bossy-Wetzel E, Goldstein JC, Fitzgerald P, Green DR: p53 inducesapoptosis by caspase activation through mitochondrial cytochrome c release.J Biol Chem 275:7337^7342, 2000

ShenY,White E: p53-dependent apoptosis pathways. Adv Cancer Res 82:55^84, 2001

LETTERS TO THE EDITOR 1209VOL. 119, NO. 5 NOVEMBER 2002