Cisplatin preferentially binds to DNA in dorsal root ganglion neurons in vitro and in vivo: a potential mechanism for neurotoxicity
Introduction
Cisplatin is a chemotherapeutic drug used as a first line treatment for metastatic ovarian and testicular cancers. A major dose limiting side effect of this drug is neurotoxicity. About 20% of patients are unable to complete a full course of cisplatin therapy due to sensory neuropathy. The mechanism of neuronal injury is unknown, but we have shown that cisplatin causes apoptosis of dorsal root ganglion (DRG) neurons in vitro and in vivo (Fischer et al., 2001, Gill and Windebank, 1998). Cell death can be prevented or retarded by increasing NGF concentration in vitro from 5 ng/ml (0.192 nM) to 100 ng/ml (3.84 nM) (Gill and Windebank, 1998).
In cancer cells, the amount of DNA crosslinking has been correlated with cisplatin cytotoxicity (Oshita and Eastman, 1993, Pera et al., 1981, Zwelling et al., 1979, Zwelling et al., 1981). Decreased platinum–DNA adduct formation correlates with cellular resistance (Yang et al., 2000). Enhanced platinum adduct repair has been shown to be a mechanism of resistance to cisplatin-mediated death in human and hamster ovarian carcinoma (Lee et al., 1993, Masuda et al., 1988), human testicular cancer (Hill et al., 1994), human head and neck cancer (Yang et al., 2000) murine leukemia (Eastman and Schulte, 1988, Sheibani et al., 1989), and rat colon adenocarcinoma (Oldenburg et al., 1994).
Reduced ability to repair DNA correlates with cisplatin sensitivity. When p53 function was disrupted in breast cancer cells, they had a reduced capacity for nucleotide excision repair (NER) which correlated with an increased sensitivity to cisplatin (Fan et al., 1995). Cisplatin has been very effective in the treatment of testicular cancers. Testis cancer cells, with a defective NER xeroderma pigmentosum group A (XPA) protein, are hypersensitive to cisplatin. They also have a reduced capacity to repair platinum–DNA adducts (Koberle et al., 1999). Testis tumor cells have been found to repair platinum adducts less efficiently than more resistant bladder cancer cells (Koberle et al., 1997). Further, inhibition of DNA repair in a cisplatin resistant cell line correlates with increased cytotoxicity of cisplatin (Masuda et al., 1988).
Previous studies have shown a correlation between adduct formation in peripheral blood cells and patient tumor response to cisplatin or cisplatin/carboplatin therapy (Dabholkar et al., 1992, Reed et al., 1987, Reed et al., 1986, Reed et al., 1993). Using an in vivo model which assumed platinum removal in peripheral leukocytes to be similar to that of a squamous cell carcinoma, Parker et al. (1993) showed that the ability of the leukocytes to repair platinum adducts correlated with the resistance of the patients' solid tumor to cisplatin treatment. In a leukemia cell line, cisplatin resistance was proportionate to a reduction in platinum–DNA cross-links compared to the parental non-resistant line. Platinum DNA–protein crosslinking was similar in both the parental and resistant lines (Zwelling et al., 1981). Some investigators have found that platinum–DNA adduct levels do not correlate with survival (Fisch et al., 1996). However, the weight of evidence demonstrates that the amount of platinum-adducted DNA does correlate with the cytotoxicity of cisplatin.
A central question is: why are differentiated neurons susceptible to a DNA damaging agent? We have found that the LD50 of cisplatin in pheochromocytoma (PC12) cells (2 μg/ml after 48 h treatment) is twice that for DRG (1μg/ml after 48 h). We demonstrate that platinum adduct formation is much higher in DRG neurons than in the rapidly dividing PC12 cell line. This difference in adduct formation is not due to the non-cycling state of DRG, as differentiated and dividing PC12 cells showed similar adduct formation. DRG neurons accumulated platinum at a higher rate than PC12 cells. Even though platinum adduct repair is perhaps the most common cause of cisplatin resistance, we did not find a significant difference in adduct repair.
We also investigated the mechanism for NGF-mediated protection from cisplatin toxicity. This could involve less platinum binding to DNA. Alternatively, the programmed cell death response might be inhibited after the formation of platinum–DNA adducts. We determined whether NGF decreased binding of cisplatin to DNA. Our results show for the first time that NGF does not alter platination of DNA, indicating that it interrupts the cisplatin-induced death pathway after adduct formation.
Section snippets
Cell culture
DRG were microdissected from the spinal cords of Harlan Sprague–Dawley rats (Madison, WI) on embryonic day 15. Ganglia were dissociated by trituration through a fine-bore glass pipette after treatment with 0.25% trypsin in L15 medium for 30 min and plated on poly-l-lysine-treated (Sigma-Aldrich, St. Louis, MO) plastic dishes. Cells were incubated at 37°C with 5% CO2 in MEM medium supplemented with 15% calf serum (Hy-Clone Laboratories, Logan, UT), 0.033 M (0.6%) glucose, 0.28 mM (50 μg/ml)
Results and discussion
Cisplatin binds to primary neuronal DNA in vitro in a way that is both dose- and time-dependent (Figs. 1a and b). Platinum was virtually undetectable in DNA from untreated cells. In DNA from DRG neurons treated with transplatin (a biologically inactive isomer of cisplatin), platinum content was 37–47% that of the cisplatin treated cells at all measured time points (24, 48, and 72 h). The gap between cisplatin binding to DRG and PC12 DNA increased dramatically over time because the PC12 platinum
Acknowledgments
The help of Steve Eckdahl and the Mayo Metals Laboratory is greatly appreciated. We also thank Gregory J. Gores, MD, Scott H. Kaufmann, MD, PhD, Paul J. Leibson, MD, PhD, and Emanuel E. Strehler, PhD, for helpful discussions and critical review of this manuscript. The secretarial and editorial assistance of Ms. Jane M. Meyer is gratefully acknowledged.
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Genomic DNA platinum content of cultured embryonic DRG neurons and PC12 cells was assayed using inductively coupled plasma mass spectrometry (ICP-MS). Throughout these studies, “cisplatin” refers to the specific drug; “platinum” to the bound form of the drug that is measured in ICP-MS.