ANTICANCER RESEARCH 28 : 2637-2640 (2008)

Dys-synchronous Regulation of XPC and XPA in Trigeminal Ganglion Neurons following Cisplatin Treatment Cycles O’NEIL W. GUTHRIE

Department of Communication Science and Disorders, University of Pittsburgh, Pittsburgh, PA 15260; Department of Otolaryngology, University of Pittsburgh, 107 Eye & Ear Institute, Pittsburgh, PA 15213; Department of Biology, Developmental, Cell and Molecular Biology Group, Duke University, French Family Science Center, Durham, NC 27708, U.S.A.

Abstract. Background: Cancer cells may survive cis- neurotoxicity also increases with increasing treatment cycles diamminedichloroplatinum-II (cisplatin) DNA damage (4). Treatment-induced neurotoxicity is relevant to all through synchronous mobilization of DNA repair proteins patients, but is particularly insidious to pediatric patients such as xeroderma pigmentosum C and A (XPC and XPA). who may suffer with developmental effects (4). The However, non-cancerous neuronal cells exhibit hyper- mechanisms that underlie cisplatin-induced peripheral vulnerability to cisplatin which is manifest as peripheral neurotoxicity are currently unresolved. However, recent neurotoxicity. The purpose of the present study was to studies have revealed that systemic cisplatin treatment evaluate the effect of cisplatin on the immunolocalization of precipitates platinated DNA adducts among the peripheral XPC and XPA in the trigeminal ganglion. Materials and neurons (ganglion cells) (5). The anticancer effect of Methods: Fischer344 rats were treated with two cycles of cisplatin is predicated on the formation of platinated DNA cisplatin (2 mg/kg/day, i.p.) and tissues were harvested for adducts (6, 7). Therefore, as in cancer cells, cisplatin may immunohistochemistry after each treatment cycle. Result: XPA induce peripheral neurotoxicity in part, by targeting DNA. immunoreactivity was evident after each treatment cycle, Cancer cells have been shown to mobilize DNA repair however, XPC immunoreactivity was suppressed following the pathways such as the nucleotide excision (NER) pathways second treatment cycle. Conclusion: The hyper-vulnerability in response to cisplatin-induced DNA adducts (8). NER is of peripheral neurons to cisplatin chemotherapy may relate partitioned into two pathways. One pathway repairs to the dys-synchronous regulation of XPC and XPA. platinated adducts among transcriptionally inactive DNA (~70% of the genome) and is called global genomic (GG) Cis-diamminedichloroplatinum-II (cisplatin) is one of the NER (9). The rate-limiting step in this pathway is adduct three most widely used anticancer agents in the world and localization which is dependent on xeroderma pigmentosum commands annual sales of $500 million (U.S.) (1, 2). It has cross-complementing protein C (XPC). XPC exhibits high become a primary agent in the treatment of head and neck binding affinity ( KD ~1-3 nM) for platinated adducts and squamous cell carcinoma, testicular cancer, small cell lung once bound it initiates the GG-NER pathway (10). The cancer, ovarian cancer and a variety of childhood carcinomas other NER pathway repairs platinated adducts on (3). Τ he anticancer activity of ciplatin increases with transcriptionally active DNA (~30% of the genome) and is increasing treatment cycles, unfortunately peripheral called transcription coupled (TC) NER. The rate-limiting step in this pathway is DNA damage localization which is dependent on the RNA polymerase II transcription complex during transcription (11). When damaged DNA is localized Correspondence to: O’neil W. Guthrie, Duke University, Department by the transcription complex, the NER protein xeroderma of Biology, Developmental, Cell and Molecular Biology (DCMB) pigmentosum cross-completing protein A (XPA) is Group, French Family, Science Center, Science Drive, Rm 4337, sequestered to the site of damage and stays bound until the P.O. Box 90338, Durham, NC 27708-0338, U.S.A. Tel: + 1 919 724 7465, Fax: +1 919 613 8177, e-mail: [email protected] damage is removed (12). XPA also plays a role in the GG- NER pathway. For instance, although XPC may initially Key Words: Cisplatin, trigeminal ganglion, nucleotide excision locate platinated adducts, the adduct cannot be removed repair. until XPA arrives (13). Both XPC and XPA have no known

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Figure 1. Repeated cisplatin treatment cycles suppress XPC expression among trigeminal ganglion neurons. (A) No immunoreactivity wa s found in the trigeminal ganglia after the immunohistochemistry procedure with the anti-XPC antibody omitted (negative control). (B) Under normal conditions (vehicle control) XPC wa s expressed in the trigeminal ganglia. (C) After the first cisplatin treatment cycle, there was still XPC expression in the trigeminal ganglia. However, after the second treatment cycle (D) and after the four day recovery period (E) XPC expression was suppressed. Scale bars =10 μ.

cellular function beyond DNA repair (14). XPC only 0.9% NaCl in 50 mM phosphate buffer, pH 7.3), followed by functions in the GG-NER pathway while XPA is required periodate-lysine-paraformaldehyde (PLP) fixative. The heads for both GG-NER and TC-NER (9, 11). Cellular survival were removed, skinned, post-fixed and decalcified in 10% formic acid then neutralized in sodium sulfite. The heads were then from cisplatin intoxication requires the cooperative trimmed, paraffin embedded, serial sectioned horizontally with a response of both pathways. The purpose of the present microtome at 8 μm and mounted on subbed slides. The animal study was to evaluate the effect of cisplatin on the care and use were approved by the Institutional Animal Care and expression of XPC and XPA in trigeminal ganglion neurons Use Committee (IACUC) of the University of Pittsburgh, through immunohistochemistry. Pittsburgh, PA, and conformed to the National Institute of Health (NIH) Guidelines for the Care and Use of Laboratory Animals. Materials and Methods Both IACUC approval and NIH guidelines ensure ethical treatment of each animal and that all attempts were made to minimize both animal use and suffering. Animal procedures. Two-month-old albino Fischer344 rats (140- 160 g) were acquired from Charles River Laboratories, Malvern, PA, USA. Cisplatin was purchased from Sigma (St. Louis, MO, Immunohistochemistry. The sections were de-paraffinized and USA) and dissolved in sterile physiological saline (pH 7.4) to a treated for 10 minutes with 30% H 2O2 in dH 2O and then rinsed concentration of 0.1 mg/ml. This stock solution was sequestered thoroughly with dH 2O. They were then heated for 20 minutes at from light and stored at 5˚C. Fifteen Fischer344 rats were treated 90-98˚C in a sodium citrate-citric acid buffer (antigen retrieval) with 2 mg/kg, i.p. of cisplatin for four days. On the fifth day, five and then rinsed thoroughly with PBS. Afterwards, the sections animals were sacrificed while the remaining ten rested for 10 were pre-treated with a blocking solution of normal horse or goat days. After this rest period, the animals were given another four- serum, 10% Triton ® X-100 and 2% bovine serum albumin day treatment of 2 mg/kg, i.p. On the 19th day, five animals were (BSA; Sigma) in PBS for 1 hour. The primary antibodies were sacrificed while the remaining five were allowed to recover then diluted in the blocking solution at a 1:200 concentration. The sacrificed on the 22nd day. A cohort of 15 Fischer344 rats anti-XPC and anti-XPA antibodies are commercially available receiving saline (vehicle; 2 mg/kg/day, i.p. ) instead of cisplatin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and served as control and were similarly sacrificed on days 5, 19 and have been characterized previously (15-17). For negative 22. Rats from each group (cisplatin and saline vehicle) were controls, the primary antibody was omitted. After 24 hour sacrificed with a lethal dose of pentobarbital (100 mg/kg, i.p. ) incubation at room temperature with the primary antibodies, the and perfused transcardially with phosphate-buffered saline (PBS; sections were rinsed with PBS. They were then treated with a

2638 Guthrie: Dys-synchronous XPC and XPA

Figure 2. Repeated cisplatin treatment cycles do not suppress XPA expression in the trigeminal ganglia. (A) No immunoreactivity wa s found in trigeminal ganglia after the immunohistochemistry procedure with the anti-XPA antibody omitted (negative control). (B) Under normal conditions (vehicle control) XPA wa s expressed in the trigeminal ganglia. After the first (C) and second (D) cisplatin treatment cycles there was still XPA expression in the trigeminal ganglia. Additionally, XPA expression was present after the four day recovery period (E). Interestingly, satellite cells exhibited prominent immunoreactivity (see arrows). Scale bars =10 μ.

biotinylated secondary antibody (anti-goat or anti-rabbit; Vector Suppressing XPC while maintaining XPA suggested that Laboratories, Temecula, CA, USA) diluted 1:500 in PBS +2% trigeminal ganglion neurons preferentially mobilized the TC- BSA for one hour at room temperature. The sections were then NER pathway over the GG-NER pathway. This suggestion rinsed in PBS, incubated with Vectastain ABC reagent (Vector is consistent with in vitro DNA repair experiments that have Laboratories) for one hour, rinsed again with PBS and then treated with a solution of Trizma pre-set crystals (1.58 g; demonstrated that the GG-NER pathway is deficient among Sigma). After this the sections were washed in PBS and then neurons (20). The mechanisms that underlie neuronal stained for 10 minutes with 3.3’-diaminobenzidine deficiency of GG-NER is unresolved (21). The current in tetrahydrochloride (DAB staining). vivo trigeminal ganglion data suggested that a dys- synchronous response between XPC and XPA may Results and Discussion participate in neuronal GG-NER deficiency. Additionally, it is possible that neuronal GG-NER deficiency may contribute NER promotes neuronal survival from endogenous mutagens to the peripheral neurotoxicity experienced by patients (18, 19). Indeed, as shown in Figures 1B and 2B, the NER undergoing repeated cisplatin treatment cycles. proteins XPC and XPA were present in the neurons under Satellite (peripheral support) cells of the trigeminal ganglia normal conditions. The present study revealed for the first time have previously been shown to be more sensitive to cisplatin that systemic cisplatin treatment cycles resulted in a dys- than neurons (22). In the current study, the satellite cells of synchronous response between XPC and XPA in the trigeminal the trigeminal ganglia showed deficient GG-NER by being ganglia. After a single cisplatin treatment cycle (2 mg/kg, i.p. immunopositive for XPA, but not XPC (Figures 1 and 2). for 4 days), both XPC and XPA were expressed in the Unlike the neurons, the satellite cells were not immunopositive trigeminal ganglia (synchronous response; Figures 1C and 2C). for XPC at any of the investigated time-points which may However, after a second cycle, the XPC expression was contribute to their hyper-sensitivity to cisplatin compared to suppressed while the XPA expression was not (dys- neurons. Therefore, deficient GG-NER (which repairs ~70% synchronous response; Figure 1D and 2D). After a four-day of the genome) among neurons and satellite cells may recovery period, the XPC expression was still suppressed while contribute to cisplatin induced peripheral neurotoxicity. The the XPA remained expressed in the trigeminal ganglia (dys- current study provides a basis for further explor ation of the synchronous response; Figure 1E and 2E). significance of GG-NER in cisplatin chemotherapy.

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