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Cellular and Molecular Neurobiology [cemn] Cellular and Molecular Neurobiology, Vol. 23, No. 2, April 2003 ( C 2003) Rapid Communication
A Novel Method of Eliminating Non-Neuronal
Proliferating Cells From Cultures of Mouse
Dorsal Root Ganglia
Parker L. Andersen,1 J. Ronald Doucette,2 and Adil J. Nazarali1,3
Received November 5, 2002; accepted December 18, 2002 1. We hypothesized that non-neuronal cells could be eliminated from primary dorsal root ganglion (DRG) cultures by including a DNA topoisomerase inhibitor (camptothecin)during culture.
2. Exposure to 20 µM camptothecin for 48 h, beginning at 3 days in vitro, reliably eliminates proliferating non-neuronal cells.
3. Following camptothecin treatment, neurons survived and continued to extend neu- rites for several weeks without obvious defects in morphology or viability.
4. Transient camptothecin exposure is therefore an efficient and fast-acting method to KEY WORDS: DRG neuron; Schwann cell; fibroblast; camptothecin culture.
INTRODUCTION
Unlike neurons of the central nervous system, neurons derived from the dorsal rootganglia (DRG) can be cultured from embryonic (Wood and Bunge, 1986; Woodand Williams, 1984), postnatal (Horie and Kim, 1984), and adult (Scott, 1977) ro-dents. Because these neurons exhibit great regeneration activity both in vivo andin vitro, and are capable of being myelinated in vitro (Bunge and Wood, 1987; 1 Laboratory of Molecular Biology, College of Pharmacy and Nutrition, University of Saskatchewan, 2 Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, Saskatchewan, 3 To whom correspondence should be addressed at Laboratory of Molecular Biology, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5C9;e-mail: [email protected]
0272-4340/03/0400-0205/0 C 2003 Plenum Publishing Corporation Cellular and Molecular Neurobiology [cemn] Andersen, Doucette, and Nazarali
Devon and Doucette, 1992; Wood and Bunge, 1986; Wood and Williams, 1984),they serve as an excellent experimental model system. One disadvantage when us-ing these cells is that the cell cultures are routinely contaminated with Schwanncells and fibroblasts, which makes experimental interpretations difficult. Existingmethods to purify DRG neurons usually involve introducing antimitotic agents suchas cytosine arabinoside or fluorodeoxyuridine into the culture medium (Masukoet al., 1979; Wood and Bunge, 1986). However these agents are often required forprolonged exposure and thus have limited success. Alternately, DRG neurons canbe partially purified because of their unique large size using Percoll density cen-trifugation (Goldenberg and De Boni, 1983). This method has the disadvantage ofa low cell yield, likely due to selection of the larger neurons and partial loss of thesmaller neurons normally residing in DRG. Furthermore, the contaminating satel-lite cells tightly associated with the neuronal somas (from which some Schwanncells are derived) have a tendency for uncontrolled proliferation. A method toquickly and reliably produce pure cultures of DRG neurons would significantly aidstudies in determining the molecular mechanisms of regulating myelinating pheno-type by Schwann cells, olfactory ensheathing cells, and cells of the oligodendrocytelineage.
One possible method to eliminate contaminating cells would be to specifically inhibit some activity essential for proliferating cells but which is not essential for thepostmitotic neurons. DNA topoisomerase 1 (TOP1) is an important DNA metabolicenzyme and a molecular target of antitumor drugs (Desai et al., 1997). TOP1 enzymeregulates DNA supercoiling by controlling strand cleavage. The enzyme cleaves asingle strand of the duplex DNA, relaxing the DNA supercoil, while the complemen-tary DNA strand is rotated before religation (Wang, 1985). This “breakage–reunion”reaction by TOP1 relaxes torsional stress generated by the progression of the tran-scriptional/replication machinery along the DNA double-helix. Camptothecin (CPT)is a plant alkaloid that inhibits TOP1 activity by blocking the religation step of thecleavage/religation reaction of TOP1 (Liu et al., 2000) progressing to DNA doublestrand breaks during DNA synthesis (Ryan et al., 1991). The alkaloid leaves the celldeficient in TOP1 (Beidler and Cheng, 1995) inducing TOP1 downregulation viathe ubiquitin/26 proteasome pathway (Desai et al., 1997, 2001). The primary mecha-nism of cell death induced by CPT is S-phase-specific (Liu et al., 2000). Small dosesof CPT stall proliferating cells in S-phase, whereas higher doses induce apoptosis(Kaufmann, 1998; Morris and Geller, 1996). However, cortical neurons are sensitiveto higher camptothecin doses resulting in apoptosis, which may correlate with theirtranscriptional activity and level of TOP1 protein expression (Morris and Geller,1996).
We hypothesized that transiently exposing primary cultures of DRG neurons to CPT would have damaging effects on DNA replication of the con-taminating Schwann cells and fibroblasts, causing their death while simultaneouslyhaving little or no effect on the postmitotic DRG neurons. Our experimentsdemonstrate that CPT treatment can serve as a rapid and reliable method toeliminate proliferating cells from DRG cell cultures without affecting neuronviability.
Cellular and Molecular Neurobiology [cemn] Eliminating Non-Neuronal Proliferating Cells From Mouse Dorsal Root Ganglia Cultures
MATERIALS AND METHODS
All chemicals were obtained from Sigma, Oakville, ON, unless otherwise speci- fied. Routine cultures of mouse DRG were obtained from newborn CD1 mice bredon the University campus. Briefly, newborn CD1 mice were killed by decapitationand submerged in 95% ethanol for approximately 2 min. DRG were aseptically re-moved and meticulously cleaned of rootlets and connective tissue. The tissue wasdisaggregated by incubating in L15 nutrient mixture (pH corrected to 7.4) contain-ing 0.1% collagenase for 90 min at 37◦C with light trituration every 30 min. Followingdigestion, horse serum was added to 10% and the cell suspension centrifuged at lowspeed for 8 min. The cell pellet was resuspended in complete growth medium con-sisting of 10% horse serum and 50 ng/mL NGF (Cedar Lane Laboratories, Canada)in DMEM (4500 mg/mL glucose and NaHCO3 reduced to 2 g/L). Cells were seededonto surfaces precoated with poly-L-lysine (50 µg/mL 70,000–150,000 Da). Cultureswere incubated at 37◦C in a humidified atmosphere of 5% CO2/95% air; the mediumwas replaced routinely every 2 days. CPT dissolved in DMSO (10 mM stock) wasincluded on day 3 of the cultures to a final concentration of 20 µM for a total periodof 48 h. Sister cultures used as controls received an equivalent volume of DMSO ormedia alone.
Phase contrast images of live cultures were captured using an Olympus IX 70 inverted microscope (Carsen Group, Inc., ON) to investigate the presence of non-neuronal cells and to observe any morphological change in the neurons due to eitherCPT or DMSO treatment. To quantitate changes in neuron viability following treat-ment, cultures were established in 96-well plates at 400 nigrosine excluding neuronsper well and neuron density determined by manually counting formaldehyde fixedsister cultures before and after treatments. The results were expressed as mean ±standard deviations (SD), and statistical analysis was performed using Student’s t test.
RESULTS AND DISCUSSIONS
By 6 days in vitro (6 DIV), control DRG cultures become confluent with Schwann cells and fibroblasts (Fig. 1(A)). If the Schwann cells and fibroblasts are allowed toproliferate for extended periods, all cells lose adherence to the culture surface. Cul-tures containing DMSO alone are indistinguishable from untreated cultures (datanot shown). Following 20 µM CPT treatment initiated at day 3 of culture for 48 h,the majority of the non-neuronal cells were eliminated by 6 DIV (Fig. 1(B)) withoccasional non-neuronal cells (approximately 1 in 200 neurons at best) present. Pu-rity is dependent on removal of all connective tissue during dissection. The neuronsretained a large nucleous with smooth soma and an extensive neurite network. DRGneurons are easily identifiable by their large spherical soma, large nucleus, distinctnucleolus, long neurites, and expression of the neuronal growth associated proteinGAP-43 immunostaining (Fig. 1(C)). These are characteristics of viable neurons. Inlower-density cultures individual neurons possess extensive neurites confirming thatthe neurons remain healthy following both removal of the non-neuronal cells and Cellular and Molecular Neurobiology [cemn] Andersen, Doucette, and Nazarali
Fig. 1. Phase contrast micrographs of newborn mouse dorsal root ganglia culture (A) at 6 days in vitro
(DIV) and (B) a comparable culture at 6 DIV following 20 µM camptothecin (CPT) treatment initiated
on day 3 for 48 h. Figure 1(C) illustrates 10 DIV culture stained with GAP-43 (mouse monoclonal 9-1E12
(a kind gift from Dr D. Schreyer) followed with an Alexa conjugated anti-mouse antibody (A-11001,
Molecular Probes, Inc., U.S.A.)). Bar = 100 µm for Fig. (A)–(C).
following transient CPT exposure. The few remaining non-neuronal cells likely havenot passed through S-phase within the 48 h treatment period and therefore are likelyquiescent fibroblast cells, although their exact phenotype has not been investigated(see arrows in Fig. 1(B)).
To investigate the effects of CPT treatment on neuron viability, neuron counts were made at several time points during the culture period, before (3 DIV) andafter CPT treatment or DMSO (vehicle) treatment or medium alone (6 and 10 DIV;Table I). No significant difference in numbers of neurons was observed followingeither CPT or DMSO treatment. The major effect on the DRG cultures from CPTexposure was induced killing of the mitotic non-neuronal cells, with no observableeffect on the neurons. Initially dose and time responses were performed to maximizethe efficiency of CPT treatment. Either increasing the CPT concentration to 1 mM orexposure to 20 µM CPT initiated at the time of plating eliminated not only the non-neuronal cells but also a significant number of the neurons. Additional prolongedexposure to CPT (7 days) also diminished the neuronal count (data not shown).
Hence a dose of 20 µm CPT and exposure of 48 h initiated on day 3 of culture Table I. Quantitation of Neurite-Expressing Cells From Newborn Mouse Dorsal Root Ganglia (DRG)
Note. DRG cultures untreated (control) or treated with 20 µM camptothecin (CPT) or vehicle (DMSO).
For details see Methods. Culture was continued for 3, 6, and 10 days in vitro (DIV). Values representthe average of three wells. Standard deviation is indicated in parentheses. No significant differenceswere observed between treatments. Additional cell counts were conducted in four separate independentexperiments in triplicate, and similar findings were observed.
Cellular and Molecular Neurobiology [cemn] Eliminating Non-Neuronal Proliferating Cells From Mouse Dorsal Root Ganglia Cultures
were found to be optimal (Fig. 1(B)), whereas higher doses or longer exposureswere detrimental to the culture. Additional transient treatment with CPT can beapplied at later stages if needed to remove remaining proliferating cells withoutdetrimental effects to the neurons (data not shown). The possibility of unobservedactions by CPT, such as an effect on transcription, cannot be ruled out. However,DRG neurons remained healthy (as determined by the appearance of large distinctnucleoli, smooth soma, and neurites) for at least 21 DIV (data not shown) followingthe 48 h transient exposure to CPT.
The DNA double-strand breaks and apoptosis induced by ara C (1-β- arabinofuranosylcytosine) in sympathetic neurons is reported to be similar to that in-duced by the topoisomerase 2 inhibitors (Tomkins et al., 1994). Fetal CNSneurons have been described to be much more susceptible to either CPT (Morris andGeller, 1996) or etoposide, an inhibitor of topoisomerase 2 (Nakajima et al., 1994),than cocultured astrocytes. The susceptibility in both cases has been ascribed to ahigher transcription activity in the neurons. A direct link between CPT-induced DNAdouble-strand breaks in the G2 phase of the cell cycle of CHO cells and transcriptionhas also been described (Mosesso et al., 2000). The DNA topoisomerase 2-β has re-cently been reported to be involved in early stage cerebellar neuron differentiationby potentiating transcription of neuronal genes (Tsutsui et al., 2001).
DRG neurons from the newborn mice are postmitotic; an effect on embryonic DRG neurons was not investigated. CPT induces reversible protein-linked DNAbreaks (PLDB) which is an important step in the toxicity of CPT (Beidler and Cheng,1995). Since cells in the S-phase are significantly more sensitive to the killing effectsof CPT, it has been proposed that PLDB collide with the replication fork during DNAsynthesis to produce a double-stranded DNA (dsDNA) break, transforming the re-versible PLDB to a more permanent dsDNA break (Beidler and Cheng, 1995; Ryanet al., 1991). CPT also induces a reduction in TOP1 protein and this downregulation inTOP1 may be a resistance mechanism to avoid the toxic levels of PLDB (Beidler andCheng, 1995). In fact there is a general negative correlation between CPT-inducedTOP1 downregulation and CPT resistance in certain cancer cell lines. The breastcancer line ZR-75-1, most susceptible to CPT, was also completely defective in CPT-induced TOP1 downregulation (Desai et al., 2001). To our knowledge, expression ofTOP1 in DRG neurons has not been documented, but it is possible that the DRGexhibit effective CPT-induced TOP1 downregulation and may explain the relative in-sensitivity of postmitotic DRG to CPT even at the higher concentration used (20 µM).
In conclusion, our results demonstrate that DRG neurons are less susceptible to transient (48 h) CPT treatment than are Schwann cells or fibroblasts. This representsan important new method that can be quickly and easily used by researchers to purifyDRG neurons in vitro. The method is being used routinely in our laboratory to purifyDRG neurons in vitro in significant numbers.
ACKNOWLEDGMENTS
Supported by a research grant from the Canadian Institutes of Health Research to A.J.N and J.R.D. We also acknowledge support from the Multiple Sclerosis Societyof Canada for technical personnel.
Cellular and Molecular Neurobiology [cemn] Andersen, Doucette, and Nazarali
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