Redox biology in normal cells and cancer: restoring function of the redox/Fyn/c-Cbl pathway in cancer cells offers new approaches to cancer treatment.

This review discusses a unique discovery path starting with novel findings on redox regulation of precursor cell and signaling pathway function and identification of a new mechanism by which relatively small changes in redox status can control entire signaling networks that regulate self-renewal, differentiation, and survival. The pathway central to this work, the redox/Fyn/c-Cbl (RFC) pathway, converts small increases in oxidative status to pan-activation of the c-Cbl ubiquitin ligase, which controls multiple receptors and other proteins of central importance in precursor cell and cancer cell function. Integration of work on the RFC pathway with attempts to understand how treatment with systemic chemotherapy causes neurological problems led to the discovery that glioblastomas (GBMs) and basal-like breast cancers (BLBCs) inhibit c-Cbl function through altered utilization of the cytoskeletal regulators Cool-1/ßpix and Cdc42, respectively. Inhibition of these proteins to restore normal c-Cbl function suppresses cancer cell division, increases sensitivity to chemotherapy, disrupts tumor-initiating cell (TIC) activity in GBMs and BLBCs, controls multiple critical TIC regulators, and also allows targeting of non-TICs. Moreover, these manipulations do not increase chemosensitivity or suppress division of nontransformed cells. Restoration of normal c-Cbl function also allows more effective harnessing of estrogen receptor-α (ERα)-independent activities of tamoxifen to activate the RFC pathway and target ERα-negative cancer cells. Our work thus provides a discovery strategy that reveals mechanisms and therapeutic targets that cannot be deduced by standard genetics analyses, which fail to reveal the metabolic information, isoform shifts, protein activation, protein complexes, and protein degradation critical to our discoveries.

Systemic 5-fluorouracil treatment causes a syndrome of delayed myelin destruction in the central nervous system.

BACKGROUND: Cancer treatment with a variety of chemotherapeutic agents often is associated with delayed adverse neurological consequences. Despite their clinical importance, almost nothing is known about the basis for such effects. It is not even known whether the occurrence of delayed adverse effects requires exposure to multiple chemotherapeutic agents, the presence of both chemotherapeutic agents and the body's own response to cancer, prolonged damage to the blood-brain barrier, inflammation or other such changes. Nor are there any animal models that could enable the study of this important problem. RESULTS: We found that clinically relevant concentrations of 5-fluorouracil (5-FU; a widely used chemotherapeutic agent) were toxic for both central nervous system (CNS) progenitor cells and non-dividing oligodendrocytes in vitro and in vivo. Short-term systemic administration of 5-FU caused both acute CNS damage and a syndrome of progressively worsening delayed damage to myelinated tracts of the CNS associated with altered transcriptional regulation in oligodendrocytes and extensive myelin pathology. Functional analysis also provided the first demonstration of delayed effects of chemotherapy on the latency of impulse conduction in the auditory system, offering the possibility of non-invasive analysis of myelin damage associated with cancer treatment. CONCLUSIONS: Our studies demonstrate that systemic treatment with a single chemotherapeutic agent, 5-FU, is sufficient to cause a syndrome of delayed CNS damage and provide the first animal model of delayed damage to white-matter tracts of individuals treated with systemic chemotherapy. Unlike that caused by local irradiation, the degeneration caused by 5-FU treatment did not correlate with either chronic inflammation or extensive vascular damage and appears to represent a new class of delayed degenerative damage in the CNS.

CNS progenitor cells and oligodendrocytes are targets of chemotherapeutic agents in vitro and in vivo.

BACKGROUND: Chemotherapy in cancer patients can be associated with serious short- and long-term adverse neurological effects, such as leukoencephalopathy and cognitive impairment, even when therapy is delivered systemically. The underlying cellular basis for these adverse effects is poorly understood. RESULTS: We found that three mainstream chemotherapeutic agents--carmustine (BCNU), cisplatin, and cytosine arabinoside (cytarabine), representing two DNA cross-linking agents and an antimetabolite, respectively--applied at clinically relevant exposure levels to cultured cells are more toxic for the progenitor cells of the CNS and for nondividing oligodendrocytes than they are for multiple cancer cell lines. Enhancement of cell death and suppression of cell division were seen in vitro and in vivo. When administered systemically in mice, these chemotherapeutic agents were associated with increased cell death and decreased cell division in the subventricular zone, in the dentate gyrus of the hippocampus and in the corpus callosum of the CNS. In some cases, cell division was reduced, and cell death increased, for weeks after drug administration ended. CONCLUSION: Identifying neural populations at risk during any cancer treatment is of great importance in developing means of reducing neurotoxicity and preserving quality of life in long-term survivors. Thus, as well as providing possible explanations for the adverse neurological effects of systemic chemotherapy, the strong correlations between our in vitro and in vivo analyses indicate that the same approaches we used to identify the reported toxicities can also provide rapid in vitro screens for analyzing new therapies and discovering means of achieving selective protection or targeted killing.

Infection with an endemic human herpesvirus disrupts critical glial precursor cell properties.

Human herpesvirus 6 (HHV-6), a common resident virus of the human CNS, has been implicated in both acute and chronic inflammatory--demyelinating diseases. Although HHV-6 persists within the human CNS and has been described to infect mature oligodendrocytes, nothing is known about the susceptibility of glial precursors, the ancestors of myelin-producing oligodendrocytes, to viral infection. We show that HHV-6 infects human glial precursor cells in vitro. Active infection was demonstrated by both electron microscopy and expression of viral gene transcripts and proteins, with subsequent formation of cell syncytia. Infection leads to alterations in cell morphology and impairment of cell replication but not increased cell death. Infected cells showed decreased proliferation as measured by bromodeoxyuridine uptake, which was confirmed by blunting of the cell growth rate of infected cells compared with uninfected controls over time. The detailed analysis using novel, fluorescent-labeled HHV-6A or HHV-6B reagents demonstrated strong G1/S phase inhibition in infected precursor cells. Cell cycle arrest in HHV-6-infected cells was associated with a profound decrease in the expression of the glial progenitor cell marker A2B5 and a corresponding increase in the oligodendrocyte differentiation marker GalC. These data demonstrate for the first time that infection of primary human glial precursor cells with a neurologically relevant human herpesvirus causes profound alterations of critical precursor cell properties. In light of recent observations that repair of CNS demyelination is dependent on the generation of mature oligodendrocytes from the glial precursor cell pool, these findings may have broad implications for both the ineffective repair seen in demyelinating diseases and the disruption of normal glial maturation.

The complex identity of brain tumors: emerging concerns regarding origin, diversity and plasticity.

Elucidation of genetic and epigenetic mechanisms underlying neoplasia is one of the great success stories of modern science, but this success has not been associated with parallel improvements in the treatment of malignant tumors. One possible explanation for this failure is that the most important variables that support growth of malignancies are not yet identified. Another possible explanation, however, is that multiple variables important in neoplastic progression combine to create a level of disease complexity not taken into account by current therapeutic approaches. The study of development and neoplasia in the CNS provides some of the strongest support for the latter view--a view that, if correct, would suggest that a radical rethinking of the biology of malignancy is required if we are to make progress in the treatment of this important medical condition.

Glutamate transporter expression and function in human glial progenitors.

Glutamate is the major neurotransmitter of the brain, whose extracellular levels are tightly controlled by glutamate transporters. Five glutamate transporters in the human brain (EAAT1-5) are present on both astroglia and neurons. We characterize the profile of three different human astroglial progenitors in vitro: human glial restricted precursors (HGRP), human astrocyte precursors (HAPC), and early-differentiated astrocytes. EAAT 1, EAAT3, and EAAT4 are all expressed in GRPs with a subsequent upregulation of EAAT1 following differentiation of GRPs into GRP-derived astrocytes in the presence of bone morphogenic protein (BMP-4). This corresponds to a significant increase in the glutamate transport capacity of these cells. EAAT2, the transporter responsible for the bulk of glutamate transport in the adult brain, is not expressed as a full-length protein, nor does it appear to have functional significance (as determined by the EAAT2 inhibitor dihydrokainate) in these precursors. A splice variant of EAAT2, termed EAAT2b, does appear to be present in low levels, however. EAAT3 and EAAT4 expression is reduced as glial maturation progresses both in astrocyte precursors and early-differentiated astrocytes and is consistent with their role in adult tissues as primarily neuronal glutamate transporters. These human glial precursors offer several advantages as tools for understanding glial biology because they can be passaged extensively in the presence of mitogens, afford the potential to study the temporal changes in glutamate transporter expression in a tightly controlled fashion, and are cultured in the absence of neuronal coculture, allowing for the independent study of astroglial biology.

Characterization of A2B5+ glial precursor cells from cryopreserved human fetal brain progenitor cells.

The identification and characterization of human neural precursor cells are critical in extending our understanding of central nervous system development from model animal systems to our own species. Moreover, availability of well-characterized populations of human cells is of potential value in endeavors ranging from cell transplantation to drug screening. We have isolated a population of continuously dividing glial-restricted precursor cells from commercially available cryopreserved 18-20 weeks old fetal brain neural progenitor cells. These human glial-restricted precursor cells are A2B5(+) and do not express polysialylated E-NCAM (PSA-NCAM). They can be grown as purified populations in serum-free medium supplemented with basic fibroblast growth factor (bFGF) and can be induced to generate cells with the antigenic characteristics of oligodendrocytes and distinct astrocytic populations.

Developing concepts in neural stem cells.

Stem Cells in the Mammalian Brain: the 4th Brain Research Interactive Symposium, at the 2001 Annual Conference of the Society for Neuroscience, San Diego, CA, USA from November 8-10 2001.

Intersections between neurobiology and oncology: tumor origin, treatment and repair of treatment-associated damage.

The number of potentially intimate relationships between brain tumors and the precursor cells that contribute to normal central nervous system (CNS) development and repair now appears to be somewhat larger than would have been anticipated only a few years ago. It also appears that understanding the vulnerabilities of CNS precursor cells, and of the specific cells that they generate, might help us to reveal the biological basis for the cognitive impairment that is increasingly recognized as an adverse effect of systemic cancer therapies. Using neural stem cells as therapeutic vehicles in the treatment of brain tumors could be modified to allow repair of the damage caused by brain tumors themselves and of the neurological impairment that is frequently associated with traditional cancer treatment approaches.

Advances in the therapy of high-grade glioma at relapse: pegylated liposomal doxorubicin.

Approaches to improve the prognosis of patients with high-grade glioma, which is still grim despite combined therapy and major advances in the development of new drugs, are highly important. Chemotherapeutic agents have not consistently produced favorable results in the relapse setting so far, with tumor responses reported in a minority of cases. Among them, doxorubicin has not shown significant efficacy, despite being one of the most effective substances in vitro and in animal models. Nevertheless, encapsulation of doxorubicin using polyethylene-glycol significantly improves penetration across the blood-brain barrier. Moreover, recent clinical trials in other entities have demonstrated that similar clinical responses can be achieved using approximately half of the dose of polyethylene-glycol-liposomes compared with conventional liposomes. Considering these facts, polyethylene-glycol-liposomal doxorubicin with and without tamoxifen was evaluated within two sequential Phase II trials performed at our institution. Polyethylene-glycol-liposomal doxorubicin (Caelyx) was efficient in a reasonable number of patients with equivalent results in comparison with other successful Phase II studies. This article will discuss the literature and our own results on polyethylene-glycol-liposomal doxorubicin, with a special focus on toxicity and efficacy data raised in clinical trials on patients with high-grade glioma.