The Effect of Antimitotics on Neuronal Cell Cultures: A thorough investigation

Cell culture of neuronal tissues like spinal cord, auditory cortex, and frontal cortex all require special care. Neuronal tissue is dissected and later undergoes dissociation. After seeding onto plates or flasks, the trick is to keep them alive without exposure to too much stress and undoubtedly protecting them from contamination. Cultures must show signs of healthy neuronal cell growth and development. The overall cell health is compromised in the case of overgrowth of non-neuronal cells. Tissue that is obtained from a specific tissue type not only contains the neuronal cells that are desired, but it also contains desired adhesion growth factors; nonneuronal, glial cells. When the tissue begins to develop, neurons as well as glial cells are seen on the plates or flasks. However, sometimes there is an overgrowth of glial cells, which effects the development of the neurons. Antimitotics are used on these neuronal cell cultures to prevent the overgrowth of non-neuronal cells. Throughout this paper I will attempt to take a look at the different kinds of antimitotics and their direct and indirect effects on the neuronal cell cultures. Actual data from experimentation is not available due to contamination experienced in the lab. However, I was able to do a thorough search on different antimitotics used and the most frequently used ones. The concerns in using antimitotics are: toxicity, when to apply it, and the concentration to apply. The most frequently used antimitotics that exhibited a noticeable effect on the neuronal cultures are Cytosine Arabinoside and Fluordeoxyuridine [3]. Cytosine Arabinoside has been used more frequently than any other antimitotic, generally for periods of time up to a week and at concentrations of 10|0,M. In all cases there is an inverse relationship between the survival of neurons and proliferation of non-neuronal cells. Results suggested that antimitotics enhance neuronal survival by reducing the number of non-neuronal cells. Also it appears that proliferating non-neuronal cells are responsible for neuronal cell death by a medium factor and not by contact with the dividing non-neuronal cells. Introduction The neuronal cells in dispersed cell cultures of nervous tissue develop globular soma, send out neurites, and develop synaptic contacts [2,5]. In addition to this morphological differentiation, non-neuronal cells are also present in these dispersed cell cultures and appear to undergo mitosis for the life of the culture. Antimitotic drugs have been used in dispersed cell cultures to control the growth of these non-neuronal cells. Reports indicate that DNA-damaging treatments including certain anticancer therapeutics cause the death of postmitotic nerve cells both in vitro and vivo [20]. It is important to understand the signaling events that control this process. It has been recently hypothesized that certain cell cycle molecules may play an important role in neuronal death signaling evoked by DNA damage. A lot of anticancer therapeutics activate death processes by creating DNA damage in a manner that is frequently dependent on the cell's proliferative capacity [9]. Several reports indicate that DNA-damaging agents also activate death programs in terminally differentiated postmitotic neurons [19]. Examples include irradiation and the S phase inhibitor cytosine arabinoside (Ara-C) [19]. It is important to understand how antimitotics cause neuronal death. Evidence suggests that cell death by antimitotic agents is subsequent formation of DNA strand breaks [20]. Cell cycle components play a role in apoptotic signaling of proliferating cells induced to die by DNA damage. Observations that postmitotic neurons, like proliferating cells, are vulnerable to chain terminators, like Ara-C, lead to the suggestion that such agents evoke neuronal death by causing DNA damage. The hypothesis was raised that DNA damage may, by some means, activate elements of the cell cycle machinery in neurons that would participate in activation of an apoptotic pathway [18]. The death of sympathetic neurons exposed t6 Ara-C can be inhibited [20], It is shown within the literature that suggested treatment with antimitotics enhance neuronal survival by reducing the number of non-neuronal cells [3]. In addition it appears that proliferating non-neuronal cells are responsible for neuronal cell death by a specific factor and not by contact with the actual proliferation of the non-neuronal cells. In order for an antimitotic to be useful it must have two characteristics. First, the drug must stop proliferation of the non-neuronal cells, and second, the drugs must not be toxic to the neuron. Neurons seem to have a love-hate relationship with non-neuronal cells. Neurons and their neurites seem to grow preferentially on non-neuronal cells when cultured. These neurites appear to derive some positive influences from the non-neuronal cells even if it is just strong attachment to the substrate. However, if non-neuronal cells are allowed to grow in an unrestricted manner, they may be responsible for the specific factor, which has a negative influence on the neurons [3]. This factor is not specifically mentioned in (Burry, Richard W., 1983). The non-neuronal population in these cultures is a heterogeneous group of cells made up of mostly cells with some endothelial cells, white blood cells, and possibly some meningeal cells. In addition are some more specific cells, like: glial cells, astrocytes, Schwann cells, oligodendrocytes, and microglial cells. The use of an antimitotic may not affect portions of all these types of cells, but might act selectively on one type because it divides most rapidly. Antimitotic drugs can enhance neuronal survival in cell cultures when they are applied at the correct time, for the proper length of time and at the right concentration. To understand the success of the various drugs, the mode of action of each drug must be considered. The prime effect of the drugs is inhibition of cell division. The sites of action can be in the synthesis of the nucleotide, the replication of the DNA, or the transcription of the DNA in the daughter cell [3]. Prior to cell division, the cell will duplicate its genome by synthesis of DNA. Thymidine, which is a nucleotide used in production of DNA, is an essential part of the process. Sources of thymidine for cells include the de novo synthesis and the salvage pathway. The de novo pathway, forms thymidine from other metabolic compounds in the cell, while the salvage pathway uses thymidine present in the culture medium [3]. Fluordeoxyuridine (FdU), for example, is an inhibitor of thymidine synthetase, the enzyme in the de novo pathway [3]. For FdU to have an effect, the culture medium must not have any thymidine or cells may use the alternate salvage pathways as a source of DNA. Aminopterin and FdU are used in the culture medium without thymidine when preparing the mixture to be applied to cultures [3]. So, both FdU and aminopterin act at the level of nucleotide synthesis. Another effect of antimitotics, besides inhibition of non-neuronal cellular growth, is intraneuronal distribution of lysosomes. Results suggest that lysosome redistribution may be dependent upon a relatively slow dissociation rate constant of these antimitotic drugs from tubulin. Thus, transport of the antimitotic may occur when normal microtubule function is compromised [22]. The most rigorous glial cell reduction is obtained by pretreating cultures before Ara-C treatment (on day 2) with epidermal growth factor (EGF) 5ng/ml [21]. The decrease in glia-specific enzyme activities and protein levels, results in neuron-enriched cultures containing <5% of glial cells. In some cases, aggregates (of both mixed cells and neuron-enriched cultures) were maintained for various intervals in medium containing a depolarizing K+ concentration. The results are impaired maturation of the neuronal cytoskeleton in cultures devoid of glial cells and decreased levels of medium neurofilaments (M-NF), low neurofilaments (L-NF) and microtubule associated proteins (MAP). These findings suggest that the presence of glial cells is critical for the developmental expression and stabilization of neuronal cytoskeleton and that depolarizing concentrations of KC1 can enhance these processes. The elimination of glial cells from fetal brain cell aggregates by Ara-C treatment was found to be even more complete in cultures pretreated on day 2 with EGF (5ng/ml), owing to the adjustment of glial cell proliferation. These cultures were deprived of all oligodendrocytes and of >95% of the astrocytes normally present in mixed-cell cultures [21]. The shortage present in neuron-enriched cultures could be reversed by the chronic treatment with high potassium levels starting at culture day 9. EGF treatment alone has no apparent influence on the developmental expression of the neuronal cytoskeleton [21]. Treatment of early brain cell aggregate cultures with Ara-C eliminates a large proportion of glial cells. It is indicated by the reduction of the total DNA content, glutamine synthetase activity, cyclic nucleotide 2', 3'-phosphohydrolase activity, and glial fibrillary acidic protein (GFAP) content, <5% of the usual glial cell population persists in these so-called neuron-enriched cultures. Immunocytochemical studies showed that the few remaining astrocytes exhibit long and thick processes that stained intensely for GFAP. The reduction of the glial cell population does not seem to affect the neuronal viability. Instead, it impaired neuronal maturation. This is suggested by the reduced levels of cytoskeletal components such as H-NF, M-NF, brain spectrin, and MAP. The immunocytochemical investigations revealed a significant deficit in neuronal connectivity. The cytoskeleton on neuron-enriched cultures appeared more variable as judged from the presence of a degradation product of brain spectrin [21]. In conclusion these results suggest that telencephalon neurons require a growth modulator provided by glial cells. This is in agreement with a growing body of evidence that suggests that the formation and stabilization of axonal and dendritic process in the developing brain are under the control of glial cell deprived positive and negative growth modulators. The nature of these factors, in particular of those conveying a positive glial signal to differentiating neurons remains to be interpreted [21]. Antimitotics Neocarzinostatin (NCS) Neocazinostatin is an antineoplastic antibiotic, which consists of a protein noncovalently linked to a chromophore [23]. It has been shown to be an effective antimitotic agent in a number of systems. It is a chemotherapeutic agent that results in either apoptosis or in morphological differentiation of cells to a Schwann cell-like phenotype. Which of these effects occurs, is an intrinsic property of the cell, rather than a result of the action of NCS and its chemical relatives. Nor neural-type neuroblastoma cells uniformly undergo apoptosis, while Sor substrate adherent-type cells undergo Schwann cell-like differentiation [11]. NCS is a member of the enediyne class of anticancer drugs. Enediynes are both DNA strand-cleaving and antimitotic agents. It is difficult to determine which of these two events is the closer trigger for apoptosis and/ or differentiation [24]. The action of enediynes is irreversible, therefore it is impossible to define a critical time within which the inevitable death pathway is enacted. Reports using nonneuronal cell lines have suggested that G2-M arrest is induced by continuous exposure to microtubule-active agents, or brief cycle delay, or induced by pulsed exposure to anodynes. However, it is has been suggested that whether a cell undergoes apoptosis or differentiation in response to DNA strand-breaking, antimitotic agents are an intrinsic property of the cell, rather than a function of the proximate mechanism of action of the agent [11]. NCS has been shown to have a direct effect upon DNA. It inhibits DNA synthesis, by inducing single strand breaks in DNA almost exclusively at thymidylate and adenylate residues in vitro and by producing alkali-labile, abasic sites at cytidylate residues at AGC sequences in double stranded oligonucleotides. Its mechanism of action may involve at least three processes: oxidation of deoxyribose to a 5'-aldehyde, release of free thymine from DNA with a phosphate bridging the gap, and removal of a hydrogen at the C-l ' position [23]. The antimitotic action of NCS on neuroblastoma cells in culture induces morphological changes. In experiments involving the antimitotic and morphological effects of neocazinostatin, cells were considered to be morphologically differentiated if they fulfilled all of the following criteria: 1) extension of more than two cellular processes; 2) enlargement of the cell body by at least a factor of 2 over control cells (i.e., largest diameter of cell greater than50 |xm) [23], Neuroblastoma cells exposed to NCS assumed the morphology of mature ganglion cells by day 3 in culture. As shown in figure 1 (appendix 1), the cells developed many processes, and the somata became larger. The morphologic change was stable for the remainder of the lifetime of the cell, despite changing the medium every 4 days. No evidence for deterioration to the neuroblastoid phenotype or additional conversion to the mature phenotype was seen after day 3. The presence or absence of serum during incubation with NCS did not alter the effects of the drug upon cell morphology (data not shown for this in the article used). The relationship of the fraction of the cells in culture that undergo morphological change to the dose of NCS is shown in figure 2 (appendix 1). The change itself is an all or none phenomenon, and increasing the dose of NCS increases the likelihood that a given cell will change. A dose of 0.5 |ig/ml induces morphological change in virtually all of the cells in the culture [23]. Cells treated with NCS on day 0 and maintained in culture after their morphological maturation (medium changed to fresh medium every 4 days) die by day 14. Control cells maintained in this manner remain viable virtually indefinitely. This implies that the morphological maturation is accompanied by some degree of physiological maturation and senescence. NCS covalently modifies DNA, and onehour exposure to this antimitotic on day 0 induces visible morphological change in the cells on day 3. None of the substances that induce morphological change in neuroblastoma cells have been demonstrated to exert such a direct chemical effect upon DNA as neocarzinostatin [23]. Effectiveness however, depends upon its continued presence in the culture medium. Neocarzinostatin (0.1 |Xg/ml) decreased the growth rate of neuroblastoma cultures. The doubling time for control cultures in the experiment shown was 1.2 days, whereas the doubling time for cells treated with 0.1 |Xg/ml of NCS was 1.7 days. In three experiments, treatment with 0.1 |Xg/ml of NCS lengthened the doubling time by 37, 43, and 50% respectively. In cultures treated with 0.5 |Xg/ml, there was no change in cell number after the 1 day in culture [23]. In conclusion, NCS can induce morphological differentiation and mitotic arrest of both murine and human neuroblastoma cell lines. It is impossible to determine from the studies performed with NCS what the relationship is between these two events. If it is assumed that they are causally related to one another, it is not possible to determine which of the two events is primary. Even though we do know that differentiated neurons do not divide, it is not known whether mitotic arrest in neural crest tissues obligates differentiation to the neuronal phenotype [23]. Vinblastine Vinblastine is a microtubule disrupter, on N-type and S-type neuroblastoma cells in culture, unlike enediynes, vinblastine is an antimitotic agent without effect on DNA. Brief treatment with vinblastine is followed by recovery of microtubule structure and function [24]. Exposure of N-type cells to the antimicrotubule antimitotic, vinblastine for 15 minutes is sufficient to induce